Enzyme and Microbial Technology 25 (1999) 161171

Review

The dielectric properties of biological cells at radiofrequencies: Applications in biotechnology

Gerard H. Markxa,*, Christopher L. Daveyb
a

Department of Chemical Engineering, University of Manchester Institute of Science & Technology, Manchester M60 1QD, UK b Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DA Wales, UK Received 6 August 1998; received in revised form 3 January 1999; accepted 14 January 1999

Abstract The study of the dielectric properties of cells in the radiofrequencies is increasingly leading to new practical applications, including online techniques for biomass measurements and novel techniques for the electrokinetic separation, manipulation, and characterization of single cells. In this review, we will discuss the dielectric properties of cells and their components and the electrical techniques that use them. This will be done mainly in the context of biotechnology but some applications in medicine will also be highlighted. 1999 Elsevier Science Inc. All rights reserved.
Keywords: Dielectrics; Interfacial polarization; Dielectric spectroscopy; Electrokinetics

1. Introduction The dielectric (electrical) properties of biological cells have been studied for over a century, and have led to such discoveries as the molecular thickness of the cell membrane and the ionic nature of nerve conduction [1,2]. Today such studies are an area of intensive research that is increasingly leading to practical and commercial applications [2 4]. In this review, we will take a bottom-up approach to discussing biological dielectrics and their applications. First, some general concepts about the dielectric properties of all materials will be introduced, followed by a description of the electrical properties of the components making up cells. The strategies used to generate models of the electrical properties of whole cells will be introduced. Finally, we will highlight some of the techniques and their practical applications. 2. Dielectrics 2.1. The dielectric properties of materials When an electric eld is applied to a material, energy in the eld is either lost by frictional motion of the constituent
* Corresponding author. Tel.: 44-161-200-4394; fax: 44-161-2004399. E-mail address: mjksggm@fs1.ce.umist.ac.uk (G.H. Markx)

charge carriers and turned into heat (resistive loss), or stored by polarization of the materials components. Polarization can be due to various effects, ranging from charge accumulation at the surfaces between materials with different electrical properties (interfacial polarization) to dipole orientation and other effects [1]. Theoretically, energy storage in magnetic elds could also occur (induction), but this is negligible in most biological materials. The response of a material to an applied electric eld is described by its conductivity ( , in S/m) and permittivity ( , in F/m). The conductivity gives a measure of its ability to conduct, i.e. let charge pass through it, whereas the permittivity gives a measure of the polarizability of the material, i.e. to store charge. Permittivity is often expressed as the relative permittivity r (or dielectric constant, dimensionless), which is dened as the permittivity relative to that of vacuum ( 0 8.854 10 12 F/m): r / 0. Because the energy in the electric eld is either stored or lost, conductivity and permittivity are related. They are often expressed as the complex permittivity * and conductivity * to take advantage of this fact in theoretical calculations: * j / or, equivalently: * j , where is the radial frequency (rad/s) of the applied electric eld and j 1. For most substances, the permittivity and conductivity are only constant over a limited frequency range. The general tendency is for permittivity to fall, and conductivity to concomitantly increase, in a series of steps as frequency

0141-0229/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 0 8 - 3

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ions being attracted from the medium to the surface to form a double layer [1,8]. 2.2.2. Cell wall The wall that surrounds many cells frequently has an elaborate structure that can have different layers each with different compositions. To live, cells need to exchange small molecules with their surroundings, and most walls have a porous structure to let this occur. The wall itself contains large amounts of polysaccharides and other natural polymers, and it is the charged groups on these materials that give the wall the properties of an ion exchanger [8,9]. This, in turn, means that the electrical properties of the wall will change with the concentration of ions present in the surrounding medium [9]. 2.2.3. Membranes The membrane surrounding the cell has a lipid bilayer structure. It is extremely thin (4 10 nm in thickness) and has incorporated into it large amounts of proteins. However, the effect of proteins and water on the membrane dielectric properties is unclear. As a result, the value of the membranes relative permittivity has been given values ranging from typically 210 [5,8,12]. Transport of ions through the membrane is highly regulated by transmembrane channel proteins, and under normal circumstances, the membrane can be regarded as highly nonconducting. From patchclamp experiments it is known that it does not show any major dispersions at lower frequencies [13]. Application of large direct current (DC) or low-frequency alternating current (AC) electric elds to a cell induces a large potential drop across the plasma membrane which can cause dielectric breakdown. This rupturing of the membrane has been used to kill cells but has also found applications in electrofusion for the creation of new hybrids and electroporation for the introduction of new genetic material into cells [14,15]. Cells maintain a voltage drop of their own over the membrane which plays a role in cell energetics and transport. The inuence of this naturally occurring membrane potential on dielectric measurements is again unclear. The effect of the membrane potential on the -dispersion is most likely limited, but on the -dispersion it is potentially large [16]. 2.2.4. Cytoplasm The cytoplasm of cells is highly complicated. It contains not only large amounts of salts, proteins, nucleic acids, and smaller molecules but in many cases (e.g. eukaryotes) various membranous structures (e.g. nucleus, vacuoles) which can also affect the dielectric properties. The value of the inside relative permittivity has been attributed values ranging from about 50 to over 200 [5,17] (compare pure water 79). In most cases, however, the cytoplasm can be r approximated as a highly conducting salt solution with a large concentration of dissolved organic material. At higher frequencies, ( 20 MHz) dispersions can occur [17] that are related to the movement of small molecules and molecular

Fig. 1. Idealized spectrum of the dielectric properties of cell suspensions and tissues. The step changes in r are called dispersions and are due to the loss of particular polarization processes as frequency increases. The -dispersion is due to the tangential ow of ions across cell surfaces, the -dispersion results from the build-up of charge at cell membranes due to the MaxwellWagner effect, the -dispersion is produced by the rotation of macromolecular side-chains and bound water, and the -dispersion is due to the dipolar rotation of small molecules particularly water.

increases. These step changes are called dispersions, and each one reects the loss of a particular polarization process at increasing frequencies. Biological materials can show quite large dispersions, especially at low frequencies (Fig. 1). These are mainly caused by interfacial polarizations at the surfaces between the different materials of which a cell is composed. Reviews of the dielectric properties of cells and the different dispersions that occur have been given in the literature [1,57]. 2.2. Electrical properties of cells Except for viruses, all living matter consists of cells that have a similar structure consisting of cytoplasm surrounded by a membrane. In many cases (plants and most microorganisms), the cell is further enveloped by a cell wall. The electrical properties of these different cellular structures will now be discussed in turn, before considering how they are incorporated into dielectric models of whole cells. 2.2.1. Cell surface The surface of the cell is the rst point of contact with the environment, and its structure can be highly complicated, including, for bacteria, S-proteins, mbriae, pili, and, for animal/human cells, the membrane glycoproteins [8,9]. The surface layer is negatively charged for nearly all cells because of the predominance there of negatively charged groups (carboxylates, phosphates). This results in positive

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Fig. 2. Multishell model of a cell. The different shells represent the cytoplasm, cell membrane, cell wall, and suspending medium. Typical values for the conductivity and permittivity are: i [5]: 210 S/m; ri [5] 50 120; mem [8,23]: 10 810 4 S/m; r mem [5]: 210; wall [11,23]: 0.011 S/m; r wall [23]: 60. From these values the overall homogeneous permittivity and conductivity can be calculated. The conductivity and to some extent the permittivity of the suspending medium can be varied by the experimenter.

sidechains in the electric eld. Especially prominent are the dispersions from the rotation of water molecules, which are different for bound and unbound water [18 20]. The dielectric properties of biological macromolecules and solutions thereof have been studied extensively, and reviews have been given by several authors [18 20]. 2.3. General strategies for modeling the dielectric properties of whole cells Cells consist of layers of materials with largely different dielectric properties. These differences lead to charge accumulation at their interfaces. Interfacial effects can give rise to substantial dispersions with permittivities orders of magnitude larger than the permittivities of each component. Interfacial polarization was rst described by Maxwell [21], who modeled mixed dielectrics in terms of layers of materials. The next major advance was by Wagner [22] who modeled the dielectric properties of dilute suspensions of spheres immersed in a material with different dielectric properties. Interfacial polarizations are often called MaxwellWagner polarizations after the two major early contributors to the eld. Cells have a highly heterogeneous structure, containing many different materials with different dielectric properties. The approach usually taken to model them is to replace the particle with a hypothetical particle of known size, shape, and distribution of charges and/or materials (for example in shells). Using this model, an overall equivalent homogeneous complex permittivity * or conductivity * is calculated for the particle (see Fig. 2). This is then used to calculate the overall suspension permittivity and conductivity (dielectric spectroscopy) or particle velocity/force etc. (electrokinetics). The description of the equations that explicitly relate these homogeneous particle properties to the measurable macroscopic properties of suspended cells will be left to the sections on each dielectric technique that

utilizes them. However, one very important feature of all * * these equations is the term ( * 2 * ) which is p m)/( p m the ClausiusMossotti factor for spherical cells. (Different equations exist for nonspherical cells.) It is this factor that encapsulates the differences in the homogeneous dielectric properties of the particles * and suspending p medium * . m Much of the early work on the measurement and modeling of the dielectric properties of cells was undertaken before World War II by Cole and Fricke. After the discovery by Hober [24] that the cell was surrounded by a thin nonconducting layer, Fricke [25] developed a shell model in which a spherical cell was surrounded by a thin shell of low conductivity. This model was later extended to include multi-shell models [26] and ellipsoidal shapes [27]. Cole made major contributions to the measurement and interpretation of dielectric spectra in general (e.g. the Cole Cole plot) as well as to those of cell suspensions and tissues [28,29]. His work on the low-frequency properties of nerve tissues led directly to the elucidation of the mechanism of ion conduction in nerves and indirectly to the patch-clamp techniques that have been so successful in the investigation of nerve conduction. Important postwar contributions to the eld were made by Schwan, who has also written a number of reviews and historical overviews [6,7,30]. The simple shell models have been highly successful for the description of the -dispersion but were inadequate in describing the abnormally high conductivities and permittivities found at low frequencies, which are caused by the cell surface charge and give rise to the -dispersion. OKonski [31] explained the abnormally high conductivity that could be seen in some materials at these frequencies by introducing the concept of surface conductivity. Schwarzs solution to the problem [32] was a model in which double layer effects were used to explain the high permittivity values seen at low frequencies. Although essentially correct in accrediting the phenomena to the double layer, in detail Schwarzs model was inadequate because it failed to include diffusion effects in the double layer itself. Various models have since been published that have aimed to correct this [3335]. The cell wall has been a relatively neglected area in dielectric research, despite being of great importance in understanding the dielectric properties of micro-organisms. An exception is the excellent work by Carstensen et al. on bacterial walls [10,11]. He showed that the wall acts as an ion exchanger and that its conductivity stays relatively constant at low ion concentrations but increases rapidly if the medium ion concentration exceeds that of the wall itself. Wall conductivities can be very high, and values over 0.5 S/m are no exception [11]. Carstensens rather crude models have been expanded by van der Wal et al. [36].

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Table 1 Techniques utilizing the dielectric properties of cells Dielectric spectroscopy [3841] Electrokinetics [42] DC Electrokinetics [4345] AC Electrokinetics Electrophoresis Electro-osmosis Dielectrophoresis [3,42,46] Electro-orientation [42] Electrorotation [42,47] Travelling wave devices [4850]

3. Dielectric techniques We will now review the different techniques that make use of the dielectric properties of cells. We will distinguish between the measurement of the dielectric properties of cell suspensions and tissues using dielectric spectroscopy (impedance spectroscopy) and electrokinetics as outlined in Table 1. A comparison of dielectric spectroscopy and AC electrokinetics of cells, and the equations used therein, has been given by Wang et al. [37], showing the importance of the ClausiusMossotti factor (Eqs. (27)) in the mathematical description of the different phenomena. 3.1. Dielectric spectroscopy Dielectric spectroscopy is the direct measurement of the electrical properties of a material by registering the relationship between the current and voltage (magnitude and phase) for a set of electrodes containing the material under investigation. A wide variety of techniques are used [20, 38 41], but a basic measurement consists of measuring the admittance domain capacitance (C, in F) and conductance (G, in S) of a block of material as a function of the applied eld frequency. One then calculates the relative permittivity and/or conductivity by using the equations r CK/ 0 and GK, which use a cell constant K (in m 1) to allow for electrode geometry. The resulting dielectric spectra (Fig. 1) are then analyzed to obtain information on the material investigated. The usual approach is to t a dispersion (e.g. a -dispersion) to the ColeCole equation [29] by using nonlinear least squares:
r r

1
1

sin

sin /2

/2
2 r

(1)

Fig. 3. ColeCole analysis of dielectric spectra. (A) The dots are hypothetical dielectric dispersion data (e.g. for a -dispersion) that have been tted to Eq. (5) (solid line) to get the best t estimates of the dispersions and r (dielectric increment), fc (characteristic frequency), ColeCole (dimensionless) r (high-frequency permittivity) values. The ColeCole can take values between 0 and 1 and is supposed to reect a distribution in relaxation times, although this has not actually been proven [5]. Typical values [5,39] for the ColeCole of the -dispersion are 0.1 0.2. Also shown are the two frequencies (f1 and f2) that can be used to estimate biomass concentration. (B) ColeCole plot of two overlapping dispersions. The hypothetical experimental dispersion data are plotted as the imaginary ( r ) versus real parts ( r ) of the permittivity to give the curve draping the two semicircles. r is calculated by using ( L)/ 0, where L is the DC ionic conductivity of the suspension. To deconvolve the data, one must t two non-overlapping semi-circles to the experimental curve as shown, one for each of the underlying dispersions. The lower frequency dispersion is to the right (labeled 1), the higher frequency one to the left (labeled 2). The arrows on the experimental data and two dispersion arcs indicate the direction of increasing frequency and the squares the values at one particular frequency. If the ColeCole of a dispersion is 0, then its semicircle will have a depressed center (dots), the angle between the center and the points on the semicircles intersection with the x axis giving an angle of /2 radians. The distance between the two x-axis intersection points gives the r, and the peak of the semicircle corresponds to the data at the dispersions fc.

or by analyzing the dielectric spectra by using a ColeCole plot (Fig. 3A and B). Once the key features (e.g. r) of the spectrum have been extracted, one must relate them to the composition of the cell suspension itself by using a relevant suspension model. For low-particle concentrations (volume fraction P, dimensionless), Eq. (2) has been derived by Wagner [22] to describe the complex permittivity of a suspension of spher-

* ical particles s with homogeneous dielectric properties * p in a homogeneous suspending medium with permittivity * . m

* s

1 P * m 1 P

* p * p * p * p

* m * 2 m * m * 2 m

(2)

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Pauly and Schwan [6,51] calculated the equivalent homogeneous dielectric properties * of spherical cells by assump ing that the cytoplasmic core was surrounded by a nonconducting membrane shell that was thin compared with the cell diameter. On insertion into Eq. (2) and simplication [6,51,52], it was found that under certain circumstances, the change in the relative permittivity ( r) of the suspension during the so-called - (interfacial) dispersion was related to the microscopic structure of the cells by r 9PrCmem/ 4 0, where Cmem is the membrane capacitance per unit area (F/m2) and r is the cell radius (m). Although not explicitly stated by Schwan and Pauly [51], 9PrCmem/4 0 r tacitly assumes that there is no wall present. This is not the case for many cells. r 9PrCmem/4 0 also only holds for spherical cells, and so if the cells are markedly non-spherical, one of the alternative models that accounts for this effect must be used [53]. These models typically assume the cells to have random orientation. If they do not, then once again the equations will fail as the permittivity and conductivity measured will become dependent on the orientation of the cells relative to the eld direction. This effect becomes particularly important in tissue measurements where one nds, for instance, erythrocyte alignment in owing blood [54] or tissue composed of cells with aligned long axes (e.g. muscle) [6]. 9PrCmem/4 0 predicts a linear relationship ber tween r and volume fraction P. However, when P gets much above 0.15, the r starts to plateau because of the fact that adjacent cells disturb the eld impinging on their neighbors. Equations have been derived to allow work at high P values, and these include those of Bruggeman [55], Hanai [56], and Schwan [6,57,58]. Interestingly, even locally increased volume fractions, as for example can be seen during rouleaux formation in blood [59], can also affect measured permittivities, and this can be used to study the dynamics of aggregation processes [59]. At very high cellular volume fractions, tissue-like suspensions are obtained that offer ideal model systems to elucidate the dielectric properties of real tissues. Measurement of the dielectric properties of tissues themselves are an important area of medical research, with applications such as impedance tomography [60], diathermy, and plethysmography [61]. If one assumes that living cells (biomass) have intact plasma membranes and that in dead cells (necromass), the membranes are lysed, then r 9PrCmem/4 0 implies that one can measure viable biomass concentrations by monitoring the r of the -dispersion. The highly conducting media cells are typically found in make high phase resolution admittance measurements difcult, and double layer effects at the electrode surfaces can cause interfering electrode polarizations. However, these problems have been overcome, and reliable methods for biomass measurements in biotechnological processes are now available. The most commonly used system is that of the Aber Instruments Biomass Monitor (BM; Aber Instruments, Aberystwyth, UK) [52,6272], which employs a four-electrode system and

Fig. 4. Permittivity (dielectric) increment of the -dispersion as a function of biomass concentration for a number of cell types showing the effect of cell size on biomass measurements. Typical cell diameters were: Catharanthus roseus 50 m (unpubl.); Saccharomyces cerevisiae 5 m (unpublished data), Micrococcus luteus, 1 m (from [58]).

specialized electronics designed for reducing electrode polarization and conductivity effects, whereas the Colloid Probe developed by Hanai et al. [7376] (and marketed by Hewlett Packard) uses a noncontact technique based on the use of magnetic elds. In a typical experiment, r (and, hence, biomass concentration) is estimated by using two frequencies, one on the low-frequency plateau of the -dispersion (f1 on Fig. 3A) and the other on the high-frequency one (f2). This value is then related to biomass concentration by a calibration factor that allows for strain-dependent cell wall, shape, size, etc. The effect of cell radius on the permittivity increment is particularly substantial [5], and this is important because the size difference between small cells such as bacteria and large cells such as plant cells can be as large as two orders of magnitude (see Fig. 4). Dielectric biomass measurements have found a niche market in the measurement of the high biomass levels found in yeast pitching [62]. However, they have also been used to follow batch fermentations of bacteria [71], yeasts [65,68, 72], fungi [70], and animal [69] and plant cells [63], as well to control continuous cultures similar to the turbidostat (permittistat) [65]. Permittistatic culture has also been used to detect chaotic growth in continuous cultures of yeast [66]. The dielectric method is relatively insensitive to noncellular material and thus allows for measurement in immobilized cell systems [68] as well as solid-state fermentations [70]. The dielectric properties of nonviable (dead) cells are often signicantly different from those of viable (live) cells because they have no intact membranes and the internal conductivity has decreased. As a result, the dielectric method can be used to follow cell death in suspensions after chemical [67], hydrodynamic [64], or other stresses.

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Fig. 5. Outline of the main electrokinetic phenomena exhibited by particles in electric elds. Electrophoresis occurs in DC electric elds, whereas AC electric elds can be used to observe the other effects. To observe electrorotation and particle movement in traveling wave devices, phase-shifted electric signals are applied to the electrodes.

3.2. Electrokinetics Electrokinetics is the movement of particles in electric elds [4,42] (Fig. 5) and includes DC effects such as electrophoresis, as well as AC effects (Table 1). Besides being used for manipulating and separating cells, electrokinetic techniques have also found utility for cell characterization. Whereas dielectric spectroscopy has been limited to measurements on suspensions containing large numbers of cells, electrokinetic techniques have the advantage that they can be used for measurements on single cells. This, in turn, means that nonlinear effects due to cell interactions at high cell concentrations are eliminated. 3.2.1. DC electrokinetic techniques We will touch only lightly upon DC electrokinetic techniques and instead refer to the literature [43 45]. Electrophoretic separation of cells [45,77] tends to be difcult because of convection currents generated at the electrodes. Approaches to overcome this include cooling [45] and the use of capillary electrophoresis [77]. The selective movement of cells in miniaturized devices by DC electric elds has been demonstrated [78]. 3.2.2. AC electrokinetic techniques To observe AC electrokinetic effects, high eld strengths are needed. Such elds can be produced either by applying high voltages to large electrodes or small voltages to small electrodes. Because of the simplicity of equipment and experimental design, the latter approach is generally favored. Although electric elds at low frequencies can cause electric breakdown of the cell membrane [14,15] and kill the cells, at higher frequencies, the voltage induced across the membrane is signicantly reduced to the point that cell

culturing at energized electrodes is possible [79]. Because of the difculties of making very small electrodes over large areas, the equipment size and the volume that can be handled is usually small, and AC electrokinetic techniques tend to be used on analytical scales mainly. This is not necessarily a setback, and it is, in fact, in miniaturized devices that they will have their main impact. Several research groups in the world, in particular in the UK, Germany, Japan, and the USA, are vying to be the rst to develop an integrated system (biofactory-on-a-chip, laboratory-on-achip, uid integrated circuit, bioelectronic chip) in which single or very small numbers of cells can be manipulated, characterized, and/or sorted [80 85]. 3.2.2.1. Dielectrophoresis. Dielectrophoresis is the movement of particles in nonuniform elds [3,46]. When a particle is subjected to an electric eld, a dipole is induced in it. If the eld is nonuniform, its strength on one side of the particle is larger than that on the other, and a resultant force acts on the particle which is called the dielectrophoretic (DEP) force. The magnitude of this force (F, in N) at a given frequency in an electric eld of magnitude E (V/m), for spherical cells, is given by Eq. 3: F 2
mr 3

Re

* p * p

* m * m

E 2 rms

(3)

Re stands for the real part of. The term E2(rms) denes the average local eld strength and gradient (in V2/m3). Because the ClausiusMossotti factor can be either positive or negative, depending on the magnitude of the particles complex permittivity relative to that of the medium, the dielectrophoretic force can be either positive or negative. Fig. 6 shows the effect of medium conductivity on the DEP

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Fig. 6. Effect of conductivity on the ClausiusMossotti factor calculated for a viable (live) yeast cell. The yeast cell was modeled by using the multishell model for spherical cells, and the parameters for the calculation were taken from [23].

force [86] on a shell-bound particle with electrical properties similar to that of a yeast cell [23]. Such results clearly show that the highest positive DEP forces are obtained at the lowest medium conductivities, and in many DEP experiments, the cells are suspended in media containing low ion concentrations. In contrast, high conductivities can induce large negative DEP forces at low frequencies (around 10 kHz), and this, too, can be used to advantage in cell separation and trapping [80,86 88]. Unfortunately, at high conductivities, larger currents are needed at lower frequencies (up to 50 kHz), which puts higher demands on equipment and can result in local heating, and large forces are exerted on the cell membrane. Such effects are reduced by using high frequencies (50 MHz1 GHz) and very small electrodes [80], and DEP experiments in high-conductivity media are often limited to these. In most electrode arrangements, the eld gradient is highly dependent upon the location within the electrode system, and so the particle velocity changes with position. Only by using so-called isomotive electrode designs [46, 87], in which the electric eld gradient is constant, can one be sure that the DEP force is spatially independent. A particle also distorts the electric eld around it, and the resulting eld nonuniformity that surrounds it causes dielectrophoretic forces between the cells that lead to their mutual attraction and chain formation [89]. This so-called pearlchain formation is an important intermediate step in electrofusion [14,15]. To measure the dielectrophoretic force, a number of approaches have been taken. Direct measurement of the velocity of single cells is possible [23] but relatively cumbersome. The attraction or repulsion of cells in suspension at electrodes has been measured by using various optical techniques including image analysis [90] and absorption measurements [91,92]. A highly sensitive technique for the measurement of the dielectrophoretic force on single cells is

the dielectrophoretic levitation of cells against gravity, and important contributions to this area have been made by Kaler and Jones [42,93,94]. Although it can also be used for cell characterization, it is in trapping and cell separation that dielectrophoresis will most likely nd some major applications. Particularly promising is the combination of dielectrophoretic forces with hydrodynamic forces such as in the combined dielectrophoretic-eld ow fractionation (DEP-FFF) technique. Early attempts at cell separation employing DEP-FFF used mainly positive dielectrophoretic forces to selectively attract one particle type in a mixture [95101]. Lately, new approaches have also been used in which negative dielectrophoretic forces levitate particles against gravity above the electrodes [80,99]. Advantages of levitational DEP-FFF include less cell adhesion to surfaces and to other cells, and the ability to use higher conductivities. Dielectrophoresis has been used for the separation of live and dead (heattreated) yeast cells [80,96] and bacteria of different species [95], and the separation of human/animal cells [98 100], in particular aimed at the separation of cancer from normal cells [98 100]. Continuous separation by using dielectrophoresis [101] and the separation of submicron particles [102] has also been demonstrated. Of interest also is the device constructed by Docoslis et al. [103] in which negative dielectrophoretic forces are used to separate viable from nonviable animal cells during fermentation. 3.2.2.2. Electrorotation. Electrorotation is the rotation of particles in electric elds. Most of the early developmental work on the electrorotation of cells was done by Arnold [47]. Although it can also be observed in nonrotating electric elds [46,47], the term is generally reserved for the rotation of particles in rotating elds. Such rotating elds can be generated by applying phase-shifted electric elds to electrodes surrounding a particle (Fig. 5). When the electric eld to which a particle is subjected rotates around the particle, a resultant torque is exerted on it because of the phase difference between the applied electric eld and the induced dipole. The magnitude of the torque (in N/m) at a given frequency is given by: 4
3 mr Im * p * p * m * m

E2

(4)

where Im means the imaginary part of. When a particle rotates in a viscous liquid (of viscosity in kg/m s) the surrounding uid exerts a hydrodynamic drag force on the particle. The resultant rotation rate (in rad/s) is then dependent on the equilibrium between the drag force and the electrorotational torque and is given by:
m

Im

* p * p

* m * m

E2

(5)

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tively little studied. Biological applications of the technique may be developed for particle characterization [112114] or in the production of structured materials [46]. 3.2.2.4. Traveling waves. In traveling wave devices [48 51], phase-shifted electric elds are applied to a railway sleeper-like electrode system, resulting in the movement of particles over the electrodes in a similar fashion to the operation of a linear motor. The force (F) exerted on the particles is given by [51]: F 4
2 mr 3

A 2 0 Im

* p * p

* m * m

(6)

where is the distance between each electrode bar (in m, which also equals the bar width) and the factor A2(0) (in N/m2) incorporates the effects of electric eld strength and its nonuniformity. As in electrorotation, the nal velocity u (in m/s) of the particle is determined by the viscous drag on the particle and is given by: u
Fig. 7. (A) Dielectrophoretic and (B) electrorotation spectra of live and dead (heat-treated) yeast cells. The model used and the values for the parameters were taken from [23].

2 3

mr

A 2 0 Im

* p * p

* m * m

(7)

Both Eqs. (4) and (5) assume the cells to be spherical, but equations for nonspherical ones have also been derived [104]. The measurement of the electrorotation rate as a function of frequency gives a rotation spectrum that can be used to obtain information on the electrical properties of the particle. Electrorotation speeds can be measured manually, but automatic measurements based on image analysis [105,106] and light scattering [107] are under development. The technique has been used to characterize a wide variety of single particles. However, it is mainly limited to particles between circa 1 and 100 , as it becomes hard to observe the rotation of small particles (especially if they are spherical) while it is difcult to apply strong enough forces to overcome friction for larger ones. The technique has also been used in the measurement of the viability (Fig. 7) and the effects of chemicals on cells [108], and the techniques has clear potential in toxicology testing and drug screening. Changes in the electrorotation spectra of latex beads by the presence of viable and nonviable micro-organisms on the surface were reported by Zhou et al. [109]. Much effort is being directed toward developing electrorotation into a method for detecting small numbers of pathogens such as Cryptosporidium and various bacteria, as well as small amounts of DNA [110,111]. 3.2.2.3. Electro-orientation. Electro-orientation is the frequency-dependent orientation of elongated particles in electric elds. Although electro-orientation of cells has been known to occur for a very long time, it has been compara-

Traveling wave devices have clear potential in so-called biofactory-on-a-chip devices [84] because of their ability to manipulate single particles in predetermined directions and switch between different tracks.

4. Discussion The general area of dielectrics in biotechnology is expanding rapidly after a number of technical developments, in particular the development of equipment for measuring the dielectric properties of suspensions in highly conducting media, as well as the use of micro-fabrication techniques for the construction of micro-electrodes in the area of AC electrokinetics. It can be expected that many more applications will be found as the techniques are further developed.

Acknowledgments Dr. C.L. Davey is grateful for the support of the Wellcome Trust. Dr. G.H. Markx thanks the Nufeld Foundation.

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