Dielectrophoresis (DEP) is useful in characterization and fractionation of particles with sufficient differences in their size and/or electrical properties. Since the DEP force acting on a particle is proportional to the particle's size, many DEP fractionation techniques are successful in separating the particles according to their size. However, for some applications it is desirable to disregard the variations in size, and perform the fractionation only according to the differences in electric conductivity or dielectric permittivity of the particles. One of the techniques most suitable for such purposes is the Dielectrophoretic Field-Flow Fractionation (DEP-FFF) that exploits the balance between the upward-pushing DEP force and the downward-pulling sedimentation force (sum of gravity and buoyancy) acting in a laminar fluid flow in a shallow microfluidic channel with electrodes at its bottom [1-3], as shown in Figure 1.
For successful application, the upward direction of the DEP force must be ensured by a suitable choice of frequency of the electric field delivered to the electrodes. Differences in electric properties of the particles affect the DEP force acting on them, but not the sedimentation force, leading to differences in the vertical equilibrium positions for the particles of the same type with differing electric properties. In a channel less than a millimeter high, the flow is laminar and has a parabolic velocity profile, due to which the particles at different vertical positions have different velocities, with the particles at the vertical middle of the channel flowing the fastest, and those close to the bottom flowing the slowest.
DEP-FFF can be used for either batch or continuous fractionation (Figure 2). For batch DEP-FFF, a batch of suspended particles is injected into the chamber and pumped through the channel (the process referred to as elution); the particles closer to the vertical middle of the channel reach its output first, and others follow later. Separation of two (or fractionation of several) classes of particles is then achieved by interchanging at appropriate times the containers into which the particles are collected at the output of the channel. For continuous DEP-FFF, the chamber must have two or more outputs positioned one above another at the end of the channel. Suspended particles are pumped into the chamber continuously, flowing through the channel and exiting into different outputs according to their vertical position. The main advantage of continuous fractionation is the possibility of real-time optimization of the electric field frequency and amplitude, as well as flow rate, while in the batch mode such optimization must be performed before the actual application. The main disadvantage of continuous fractionation is the need for 3-D structures for vertical separation at the output, which are demanding to manufacture. This can be avoided by modifying the technique as to generate horizontal deflection of trajectories of different particles, which can be achieved by suitable shaping and positioning of the electrodes , or/and by introducing phase shifts between the signals delivered to individual electrodes , but such modifications generally increase the dependence of fractionation on particle size.
The general drawback of DEP-FFF chambers are their relatively large dimensions, as channel lengths of centimeters or more are required for efficient fractionation, which makes them unsuitable for micro- and nanoscale systems. Also, the electrodes are typically deposited on glass, due to which such chambers are fragile, but in cases where this is problematic, a more flexible substrate such as polyimide can be used . Above a certain frequency of the DEP-generating electric field, the DEP force changes direction and starts pulling the particles downwards (the effect of pulling the particles towards the electrodes is referred to as positive DEP, while pushing them away is negative DEP), which can be useful for particle trapping , but is generally to be avoided as the particles can attach to the electrodes.
First successful applications of DEP-FFF were reported in 1997 [1,2], and to date it is mainly used for fractionation of biological cells based on differences in their viability, phase of the cell cycle, stage of differentiation, or damage to the plasma membrane. Examples of more recent applications include separation of cancerous and non-cancerous cells , ; of viable and non-viable cells ; fractionation of different types of blood cells ; isolation of rare cells , enrichment of a putative stem cell population  and separation of electroporated cells from non-porated ones , . A fractionation of carbon nanotubes was also reported , while for fractionation of molecules, purely gravitational FFF is better choice .
For biological cell separation, DEP-FFF is a sensitive method; in practical applications, achievable separation efficiencies can exceed 90 % , , , and enrichments of rare specimen can be up to 14-fold . With robust designs and reliable protocols, it could become an established tool in laboratory bioanalysis.
|Figure 1: Representation of the gradient of |E|2 in V2/m3 (color map), the fluid velocity profile (arrows at the left), and the forces acting on particles in a DEP-FFF channel with electrode width, interelectrode gap and channel height of 100 µm, and with 3 V interelectrode voltage.|
|Figure 2: Representation of the two modes of DEP-FFF: (a) batch fractionation; (b) continuous fractionation.|