Electrophoresis with No Charge

Professor Hywel Morgan

Electrophoresis is a popular method of separation, but is limited because it can only separate charged particles. A rarely used form of electric-field based separation called dielectrophoresis does not have this limitation and opens up a whole new realm of possibilities in chromatographic analysis with the advent of ‘lab-on-a-chip’ technology. The Column spoke to Professor Hywel Morgan from the School of Electronics and Computer Science at the University of Southampton about this exciting technique.

First of all, can you tell us about the history of dielectrophoresis?
Dielectrophoresis (DEP) was first described over half a century ago by H.A. Pohl at the Oklahoma State University. He developed the first DEP-based particle characterization and separation systems using large-scale experimental apparatus. Because his systems were relatively large, he used high voltages to produce the electric fields and the forces that were needed to see movement and separation.

It was not until the 1980s that DEP became more than an idle curiosity. Researchers, particularly Ron Pethig at the University of Wales, Bangor, UK, made huge progress in this field by making smaller electrodes using micro-chip technology. This meant that experiments could be done with much lower voltages - a whole range of new phenomena were soon discovered.

Other groups, notably in Germany and the US, also developed this technology, and now dielectrophoresis is used in a wide range of applications, including nanoparticle manipulation. The interest in this technology has been accelerating: in the early 1980s there were typically a handful of papers published each year; since the beginning of 2005 there have been 136 papers published in peer-reviewed journals.

What is the principle behind this technique in separation science?
It’s best if we start by reminding ourselves about electrophoresis. Electrophoresis is a widely used technique which can separate particles according to their net charge (or zeta potential) and size in a uniform electric field. Particles (or molecules) with different net charge and sizes will move at different speeds, leading to separation over time. In electrophoresis, the electric field is generated using electrodes positioned external to the separation column. The electric field within the separation medium is uniform everywhere.

Dielectrophoresis is a technique which separates particles not on the basis of their fixed charge or zeta potential, but on the basis of a charge distribution which occurs in and around the particle when an electric field is applied. This distribution of charge creates what is called a dipole moment. In a uniform electric field, such as used in electrophoresis, this dipole doesn’t experience any net force and doesn’t move.

However, the trick is to make the electric field non-uniform in space. In other words, to design microelectrodes that produce an electric field that varies in a controlled way across the dimensionsof the system. For example, near a sharp tip, the electric field would be higher than near the flat plate.

A non-uniform field (or field gradient) means that the dipole does experience a force, and it is this force that gives rise to dielectrophoresis.

Importantly, the magnitude of the dipole around the particle (and also its direction) depends on the frequency of the applied electric field. Electrophoresis is always performed in a DC field; DEP is generally performed using an alternating current (AC) field, although DC can be used. This has two important advantages, electrolysis is avoided if the frequency is higher than a few hundred kHz, and also the frequency of the field can be adjusted for the best separation of the particles of interest.

Why is it a more viable option now?
Dielectrophoresis only occurs in an electric field that is nonuniform in space. Generating a uniform field is fairly easy, but generating a non-uniform field is rather more difficult. In the early days of DEP, large structures were machined out of metal and hundreds of volts were needed to create a significant DEP force on a particle.

Generating high voltages at DC is difficult enough, generating high voltages at frequencies of hundred of kHz is very difficult. Now, we can make very small electrodes using micro-fabrication techniques borrowed from the semiconductor industry.

Essentially, the force produced by the electric field varies with the inverse cube of the electrode gap, so that reducing the electrode gap by a factor of 10 increases the force by 1000, which is a massive gain. Nowadays, we can play all sorts of tricks with microelectrodes and simple frequency generators that work up to 10 V.

What analytes do you think this technique could be useful for? Why?
The vast majority of DEP separation and chromatography has been done on particles such as cells, bacteria and latex beads. This is because the forces produced by DEP are relatively small and also short range, in other words the particle sees a force when it is fairly close to an electrode. A significant amount of progress has been made in developing cell separation methods, notably by Peter Gascoyne’s group at the M.D. Anderson Cancer Center at the University of Texas in Houston. They developed separation technologies based on combining DEP with field flow fractionation. As we scale down in size to ever smaller particles life becomes harder and harder. The DEP force, which in the end is what leads to separation, reduces with particle volume. Large particles such as cells can be moved and separated with relative ease. When we get down to particles the size of molecules, then Brownian motion also has to be considered. Maintaining sizeable electric field gradients over large enough distances to achieve effective separation is a significant technological challenge. In principle, DEP separation should be amenable to almost any particle.

We know most about the behaviour of solid objects like latex beads; our knowledge and understanding of the dielectrophoretic properties of macromolecules (proteins) is still limited. In this context DEP has the potential to make contributions in the field of chromatography, both as a stand-alone separation technology and in tandem with electrophoretic methods.

What advantage does it offer over existing techniques?
These days it is relatively easy to design and manufacture a range of different microelectrodes into lab-on-a-chip systems. These can be used for local manipulation, collection and separation of particles within a microfluidic system. Fine tuning of the system is possible simply by adjusting the frequency or voltage, something which is not possible with electrophoresis.

Can you give an example of this technique in action?
DEP systems are being integrated as processing units with micro-fluidic systems. One particularly interesting area is in the Plot of the electric field magnitude above a castellated electrode array showing regions of maximum and minimum field corresponding to positive and negative DEP. integration of micro-electrodes into a multi-functional DEP- based cell processing chip capable of cell filtering, separation, lysis and marker molecule identification, for example, gene analysis.

Can you describe some interesting examples of this technique in action?
The technology has been used to separate, manipulate and stretch DNA in basic biophysical studies; work pioneered by the group of Washizu in Japan. DEP could be exploited in a wide range of small-scale molecular analysis and manipulation systems. We have developed methods for separating and trapping nanoparticles and viruses. We have been able to separate particles according to their surface chemistry by exploiting small changes that occur in the electrical properties of nanobeads during the binding of molecules.

DEP has also been combined with microfluidics to produce highly sophisticated cell-handling chips, with applications in many areas of biology, from high- throughput analysis and sorting to stem cell research.

DEP is even beginning to be used as a tool for the non-contact assembly of nano-scale component for electrical circuits. With all the recent interest in nano-technology, one of the most reported uses for dielectrophoresis over the last year or two has been as a tool for the separation and controlled assembly of carbon nanotubes.

What are your team working on at the moment?
We are currently pursuing two main research areas: Integration of DEP into complex microfluidic systems for highthroughput cell and particle multi-parameter analysis. This
technology has widespread application in many areas from cell biology to genomics and proteomics.

Development of new nano-particle DEP analysis and separation techniques. We are making micro- and nanoelectrode systems to try to improve our understanding of the DEP behaviour of nano-particles. The goal is the development of enhanced protein and small molecule separation/chromatographic systems. This project contains a mix of basic colloid science, numerical modelling and advanced micro- and nano-fabrication technologies.

What is the main inhibiting factor on this technique becoming more popular?
One of the main issues surrounding the use of DEP is that research groups need to have access to microfabrication facilities in order to design and manufacture microelectrodes. This increased overhead has limited the uptake of the technology in the past, although both the upfront cost and manufacture costs are being continually reduced. Perhaps the main issue is that DEP is a little more complex than electrophoresis, one has to control many parameters, frequency and voltage, electrode geometry, sample characteristics etc.

This extra layer of complexity has in the past made DEP appear to be a complicated technology; “not worth the effort!” Current trends indicate that many research groups are overcoming this inhibition and discovering that DEP is indeed a technology worth exploring further.

Research Publications » ECS Research online