Department of Chemistry & Biochemistry, Arizona State University, Tempe, AZ 85287
Analytical technologies have reached a high level of accuracy and sensitivity, but they still face limitations under extreme conditions. One challenge, for example, comprises the detection and quantification of biomarkers at the extremely low concentration limit. This analytical task becomes even more challenging as biomarkers are found in body fluids representing highly complex matrices. Another example arises from single cell studies. In order to resolve heterogeneity within cell populations, techniques are required to be sensitive enough to separate and quantify constituents of single cells. Powerful analytical techniques for many diagnostic applications demand adequate pre-concentration, separation and detection of biomolecules. Thus, for challenging analytes, hyphenated and multidimensional separation techniques often prove useful to resolve biomolecular complexity. This focus article discusses a novel mechanism for biomolecule separation, namely dielectrophoresis (DEP), and suggests adding it to the portfolio of standard bioanalytical techniques.
Probably the most commonly known electrokinetic technique is electrophoresis in which charged analytes migrate in a uniform electric field. In DEP, in contrast, a polarizable particle is manipulated in a non-uniform electric field. During DEP manipulation, polarizable particles (both charged and neutral particles) experience a force in an electric field gradient. This dielectrophoretic force is determined by the spatial changes in electric field and the induced dipole moment. The latter arises from the frequency dependent polarizability and the electric field. The resulting DEP migration of the particle is either directed to the regions of high electric field gradients (positive DEP) or away from these regions (negative DEP). It is important to note that DEP can be exploited by tuning the applied electric fields. For example, sufficiently high field gradients allow for concentration of analytes, whereas only subtle differences in DEP trapping behavior of analytes can be used for separation and fractionation.
|Figure 1: Schematics demonstrating the counter ion distribution around a charged macromolecule in solution (not to scale) without (left) and with application of an electric field (right). a) A long DNA molecule is shown in coiled conformation, b) A negatively charged protein is depicted in native conformation.|
The DEP of homogeneous particles in electrolyte solutions as well as some biological objects is well understood. For example, DEP behavior has been extensively studied for biologically relevant objects such as cells [1-6] or viruses  with applications in cytometry  as well as separation [5;9-12]. For biomolecules however, the underlying polarization mechanism is not well understood. The detailed mechanism of biomolecule polarization remains unclear and little is known on the variation of the polarization mechanism between different biomolecules. A coarse explanation can be given considering a macromolecule, such as a long DNA molecule, in an electrolyte solution. Figure 1a schematically shows a long DNA molecule (a few kbp long), which takes a coiled conformation in an aqueous solution surrounded by its counter ion cloud. Upon application of an electric field, this counter ion cloud shifts leading to polarization. It is commonly accepted that this counter ion cloud polarization is mainly responsible for the dielectrophoretic response of DNA. However, a detailed mechanism involved in DNA polarization as well as the dependence of the dielectrophoretic response on the DNA length and structure remains unclear [13;14]. Commonly, polarizabilities are found to increase with DNA length [15-19], but the reported scaling laws differ, presumably due to varying experimental conditions. Nonetheless, researchers were able to manipulate DNA in a size range from several tenths of bp to Mbp with DEP. Moreover, using DEP manipulation to separate DNA was first proposed in  and recently a microfluidic device has been developed capable of separating linear DNA by size as well as DNA topoisomers [18;21].
The examples with DNA raise the question of whether other biomolecules, such as proteins, could also be manipulated by DEP. Figure 1b demonstrates that the same polarization mechanism can be applied to other charged macromolecules such as proteins. However, the small size of proteins (nm) and presumably smaller polarizability seem to require enormous electric field gradients in order to achieve significant DEP migration or trapping. The molecular complexity and variation in proteins, on the other hand might be exploitable for DEP, in a sense that the DEP migration leads to a selective criterion for manipulation including concentration and separation. Indeed, recent experimental studies support the idea that DEP forces on proteins are sufficiently high to apply DEP for analytical applications. Experimentally, devices in which high electric field gradients can be evoked have been used to manipulate proteins by DEP. They include microfabricated electrodes , nanopipettes  and insulating arrays integrated in microfluidic channels [24;25] . Proteins well below molecular weights of 100 kDa such as bovine serum albumin  or larger, but diagnostically relevant, immunoglobulin G molecules [23;25] were successfully manipulated by DEP. In comparison to DNA however, current knowledge on protein DEP is much less developed.
The recent advances in biomolecule DEP thus suggest that DEP could indeed adjoin as a new tool in the portfolio of bioanalytical techniques. Although impressive examples on protein and DNA DEP were demonstrated, mechanistic detail has yet to be discerned. In future years, we should thus expect to see a large variety of fundamental studies on the migration and trapping of proteins and DNA, which will allow us to predict important fractionation and separation parameters, such as the applicable molecular weight range, dependence on molecular conformation and resolution. Exploiting DEP for pre-concentration could also find applications in improving detections limits of biomolecules with existing methods. Last but not least, the conjunction of DEP with other separation methods, such as linear electrophoretic methods, is envisioned to improve the resolving power of multidimensional techniques.