Manipulation of Herpes Simplex Virus Type 1 by
Dielectrophoresis.
Michael P. Hughes 1 , Hywel Morgan 1 * , Frazer J. Rixon 2 , Julian P. H. Burt3 and
Ronald Pethig3
1
Bioelectronics Research Centre
University of Glasgow
Glasgow G12 8QQ
Scotland, UK
2
MRC Virology Unit,
University of Glasgow
Glasgow G12 8QQ
Scotland, UK
3 Institute of Molecular and Bimolecular Electronics
University of Wales, Bangor
Dean Street,
Bangor
Gwynedd, LL57 1UT
Wales, UK.
Key Words: AC Electrokinetics, Dielectric properties, Nanotechnology, Particle
manipulation.
 Author to whom correspondence should be addressed, h.morgan@elec.gla.ac.uk
FAX +44 (0) 141 330 4907
1
Summary
The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian
virus, Herpes Simplex Virus, Type-1 is described. It is demonstrated that over the
range 10kHz - 20MHz, these viral particles, when suspended in an aqueous medium
of conductivity 5mSm-1, can be manipulated by both positive and negative
dielectrophoresis using microfabricated electrode arrays. The observed transition from
positive to negative dielectrophoresis at frequencies around 4.5MHz is in qualitative
agreement with a simple model of the virus as a conducting particle surrounded by an
insulating membrane.
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1. Introduction
Dielectrophoresis (DEP) is the motion of particles caused by dielectric polarization
effects in non-uniform electric fields [1-3]. Unlike electrophoresis, where the force
acting on a particle is determined by its net charge, the DEP force depends on the
volume and dielectric properties of the particle. For a spherical particle of radius r the
DEP force is given by:
FDEP  2r 3m Re f CM E 2
(1)
where  m is the absolute permittivity of the suspending medium, E is the local (rms)
electric field,  is the del vector operator and Re  f CM  is the real part of the ClausiusMossotti factor, defined as:
f CM
 p*  m*
 *
 p  2m*
(2)
In equation (2)  p* and  m* are the complex permittivities of the particle and medium
respectively, where  *   
j
and  is the permittivity,  the conductivity,  the

angular frequency of the applied field, and j =  1 . Depending on the relative
magnitudes of  p* and  m* , the DEP force acting on a particle can cause it to move
either towards or away from high-field regions at electrode edges. These two effects
are termed positive and negative DEP respectively.
DEP has been used for the separation, characterisation and investigation of
bioparticles on the micron scale, such as cells [4,5] and bacteria [6]. However, until
recently the constraints of photolithography imposed lower limits on the size of the
electrodes and thus on the magnitudes of the field E and gradient E that could be
generated using applied voltages of modest amplitude. This in turn imposed a lower
limit on the size of particles that could be manipulated. Recent advances in
microfabrication technology have now made it possible to construct electrodes with
features small enough to generate electric fields greater than 10 7 Vm 1 across gaps of
2m or less. This has allowed researchers to dielectrophoretically manipulate submicron size latex spheres, viruses and proteins [7-12].
In this paper we present evidence for the frequency-dependent dielectrophoretic
behaviour of Herpes Simplex virus type 1 (HSV-1) in microfabricated electrode
arrays. Studies by other researchers have demonstrated positive dielectrophoretic
collection at electrode edges of Tobacco Mosaic virus [12,13] and the negative
dielectrophoretic behaviour of Sendai and Influenza virus [7,8]. In this work we
demonstrate that when HSV-1 viruses are suspended in a mannitol solution of
conductivity 5 mSm-1 they exhibit positive DEP below 4.5 MHz and negative DEP at
higher frequencies. Furthermore, by determining the frequency dependence of the
rate of collection of fluorescently labelled virus particles in a DEP trap, new
3
information has been determined regarding the dielectric characteristics of the HSV-1
virus.
HSV-1 is a human pathogen best known for causing cold sores although infection can
have more serious consequences [14]. A transmission electron micrograph of a HSV1 particle is shown in Figure 1 (a). The complete particle, or virion, is approximately
200-250nm in diameter. A diagrammatic representation of a virus particle is shown in
Figure 1 (b). The viral envelope is a lipid bilayer which contains a number of
different glycoproteins. These extend outwards and form a layer approximately 1020nm thick around the envelope [15]. The envelope encloses a thick amorphous
protein gel called the Tegument, which in turn surrounds the capsid [16]. The capsid
is a robust icosahedral protein structure which contains the viral DNA. HSV-1 is one
of the largest mammalian viruses, and in electrical terms can be thought of as
resembling a scaled down version of a mammalian cell, with an insulating membrane
and a conducting core.
2. Materials and Methods
2.1 Virus Preparation
HSV-1 virions were purified using protocols established by Szilagyi and Cunningham
[17]. The virus particles were pelleted from tissue culture medium (TCM) by
centrifugation at 23,000 g for 2 hours at 4oC. They were resuspended in TCM and
centrifuged through a 35 ml gradient of 5-15% w/w Ficoll 400 in TCM at 12,000 rpm
in a Sorvall AH629 rotor. The virion band was collected, diluted in TCM, pelleted at
20,000 rpm in AH629 tubes, resuspended in an appropriate volume of TCM and
frozen at -70C for future use.
The viruses were fluorescently labelled with NBD-dihexadecylamine (Molecular
Probes Inc). The dye was dissolved in DMF at a concentration of 2 mg/ml and then
added to the virus solution (in TCM) at a 1:100 dilution. Labelling with this dye had
no effect on the infectivity of the virions. Particles were incubated for 20mins at
room temperature, pelleted at 20,000 rpm for 10 minutes in a Sorvall TLA 100.2 rotor
and resuspended in iso-osmotic (280mM) mannitol solution. For observations of the
dielectrophoretic spectra viruses were resuspended in a 280mM mannitol solution
with the conductivity adjusted to 5mSm-1 by the addition of a small amount of EDTA.
2.2 Microelectrodes
Electrode arrays based on the polynomial design [18] were used and these are shown
in figure 2. Electrodes were fabricated with a gap of 2m between nearest neighbour
electrodes and 6m gap across the centre. The arrays were made using
photolithography. A micro-litre volume chamber was constructed around the
electrode using perspex spacers and adhesives. The chamber had a depth of
approximately 50m and a sample volume of the order of 1l. Virus solution was
pipetted into the chamber and the assembly sealed with a cover slip.
2.3 Experimental
The electrodes were powered using a Hewlett-Packard signal generator providing
10Vpk-pk sinusoidal signals over the frequency range 1 kHz-20 MHz. Potentials
were applied to give a 180 phase difference between adjacent electrodes.
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Experiments were observed using a Nikon Microphot microscope, a Photonic Science
Isis II image-intensifying camera and Sony S-VHS video recorder. Conductivity
measurements were performed using a Hewlett-Packard 4192A-impedance analyser
and a Sentek conductivity cell in the range 100kHz to 1MHz. Due to HSV-1’s
infectious nature, class II containment procedures were followed at all times.
3. Results
3.1 Observation of DEP collection
Figure 3 shows three fluorescence micrographs of HSV-1 particles attracted to the
high field regions at the electrode edges under positive DEP. These images were
taken at 30 second intervals after energising the electrodes with 5V pk-pk, 500 kHz,
signals. Individual virions can be seen in the image, and these were observed to
continuously collect at the electrode edges for as long as the field was applied.
Positive DEP collection was observed at frequencies below 4 MHz (down to the
lowest frequency used of 10 kHz). At frequencies above approximately 5 MHz the
virions collected in the centre of the electrode array under negative DEP as shown in
figure 4. This figure depicts three photographs taken at 30 second intervals after
application of 5V, 10 MHz, signal. At this voltage, frequency and medium
conductivity of 5 mSm-1, the virions were observed to be levitated at least 7m above
the electrodes in a column approximately 2m diameter and 50m high. The column
of virions grew in size over a few minutes until it stabilised at these dimensions, then
virions were observed to drift out from the top of the column as new ones arrived at
the bottom. The diameter of the column remained stable once filled, as can be seen
from the time-sequences of figure 4.
Throughout the experiment, Brownian motion of particles was observed. However
the magnitude of this motion was much smaller than that due to the DEP force, and as
such did not interfere with the experiment. Interesting hydrodynamic effects were
also observed. For example, at the voltage used in these experiments and for
frequencies below about 150 kHz convection forces were found to interfere with the
DEP forces in a similar manner to that reported by Müller et al [9]. Fluid flow was
observed to occur driving particles in from the electrode edges into and along the
electrode arms. In particular at frequencies of approximately 30kHz and below,
virions were observed to collect at the centre of the electrode array in a manner
similar to that resulting from negative DEP. However, instead of being restricted in
size by the 2 m diameter field funnel formed by the electrodes, the spherical
aggregation of virions grew to a radius of 20 m or more, as illustrated in figure 5.
This aggregation effect was found to increase significantly as the applied voltage was
increased to 10V. At this voltage, virion aggregations were observed to form for
frequencies up to 120 kHz. Collection of virions at the electrode edges by positive
DEP was also maintained during this effect.
In addition to the large central accumulation of virions, small aggregations were also
occasionally observed at distinct points along the inter-electrode arms, approximately
2/3 of the distance between the centre and edge of the arrays. At these points the
aggregates on the electrodes were also levitated, and driven away from electrode
edges by streaming currents. These aggregations are illustrated in Figure 6. This
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effect was also observed to occur at the much higher frequencies of 4-5 MHz, where
the DEP force reversed polarity, from positive to negative.
3.2 Determination of Collection Rate
In order to determine the relative magnitude of the DEP force on the virions as a
function of frequency, the rate of their collection at the region of high field gradient
was determined. By determining the rate at which particles collect, an approximation
may be made to the relative force experienced by the particles. A similar method has
been used previously to determine the DEP behaviour of yeast cells by measuring the
optical absorbance of yeast solutions in AC fields [19]. Measurements were
performed by video-capturing images of the electrode array at five 30-second
intervals after the application of 5V pk-pk signal at a given frequency. The
fluorescence excitation light was blanked off between measurements to avoid
photobleaching. The captured images were converted into bitmaps and an analysis
was performed on the rate of particle collection, based on the light intensity and total
frame area illuminated (i.e. containing a fluorescing virus). The overall rate of
particle collection was determined by comparing successive frames. The mean
normalised particle collection rate in the range 100 kHz – 10 MHz is shown in figure
7. Measurements at frequencies below 100 kHz were discarded due to interference by
fluid convection effects.
4. Discussion
Fuhr and co-workers [7,8] have shown that viruses, when suspended in a medium of
conductivity 74 mSm-1, can collect as aggregates in field cages using negative DEP.
This effect was observed for frequencies up to 4 MHz, and was found to be
frequency-dependent. In this work we have shown that an enveloped virus (HSV-1)
when suspended in a medium of conductivity 5 mSm-1 can, depending on the
frequency, exhibit both positive and negative DEP.
The frequency dependent DEP behaviour of particles can be dielectrically
characterised in terms of the so-called multishell model [5]. This model can provide a
theoretical prediction of how the Clausius Mossotti factor (see equations 1 & 2) varies
with frequency. We have modelled the HSV-1 virus as a conducting particle
surrounded by an insulating membrane. It has been demonstrated [20] that the
conductivity of the lipid membrane surrounding a cell consists of a component
through the membrane  b as well as a surface component. The total conductance of

K
the membrane can be written [20,21] as: G mem  b  2 2s where K s is the surface
t
a
conductance, t is the membrane thickness and a is the particle radius. The
conductivity of an intact biological membrane is usually very low (<10-8 Sm-1) and
the surface conductance is usually of the order of 1nS [21]. For particles of the
K
diameter of HSV (250nm) the surface conductance dominates, i.e. G mem  2 2s (for
a
 b < 5 x 10-3 Sm-1). As shown in figure 8, a good fit to the experimental data occurs
6
if the surface conductance is assigned a value of 3.5nS (+/- 0.5nS), the viral interior
(tegument) is assigned a conductivity of 8 mSm-1 (+/-2 mSm-1) and a relative
permittivity of 70 (+/- 5). The fit is completely insensitive to membrane permittivity.
The interior of the virus consists of a “protein gel” and the estimated value of
permittivity could reflect the fact that this is primarily composed of water.
These parameters predict a DEP spectrum that matches the experimental data, with a
crossover from positive DEP to negative DEP occurring around 4-5 MHz.
Analysis of the collection rate of virus particles in the high field regions of the
electrodes indicates that the particles exhibit two dispersions - a low-frequency
dispersion at 200 kHz and a higher-frequency dispersion at 2MHz leading to the DEP
crossover at 4.5MHz. For frequencies above approximately 700kHz, the rate
measurement is in agreement with the simple interfacial polarisation model with the
cross over frequency at 4-5MHz. Below these frequencies it appears that the particle
experiences an increase in the collection rate, i.e. a positive DEP force which
increases with decreasing frequency, rather than a constant DEP force as predicted by
the Clausius Mossotti plot of figure 8. It is probable that the low frequency dispersion
seen in the collection rate (at 200kHz) is associated with the relaxation of the diffuse
part of the double-layer surrounding the particle. This polarisation would lead to an
increase in the low frequency polarisability of the particle and thus an increase in the
positive DEP force. Similar DEP effects, also considered to be associated with the
dynamics of the electrical double-layer, have been reported for microorganisms [22],
cells [23] and latex beads [24].
In addition to DEP forces, the movement of fluid causes additional forces on the
particles; forces which act with or against the DEP force. Owing to the scale of the
electrode designs used here, electric field strengths greater than 106 V/m can be
generated. In certain frequency and conductivity windows the high electric field
strengths give rise to local temperature gradients which in turn cause discontinuities in
the conductivity and permittivity of the liquid. These changes give rise to coulomb
and/or dielectric forces on the liquid [25,26] which can induce complex patterns of
fluid flow. At low frequencies the motion of the fluid is governed by free charge in
the medium and depends only on  and E 2 . Therefore liquid moves from regions
of high temperature (high fields at the electrode edge) to regions of low temperature
over the electrodes. In these circumstances it is the overall balance of forces
(including DEP and gravity) that determines the particle behaviour.
A 3-dimensional electric field plot for the polynomial electrode array is shown in
figure 9. This plot was obtained using the Moments method, [27] with software
running on a SPARC 20 WorkStation under Fortran 77, and data analysis was
performed using Matlab (The Mathworks, Inc). The figure is a cross section of the 3D field solution in a plane 10m above the electrodes, a point corresponding to the
approximate levitation height of the small aggregates seen in the arms of the
electrodes. The figure shows how the electric field varies in magnitude across the
whole of the electrode region in the x-y plane. It can be seen that there are 4 maxima
in field strength (at this height) at discrete locations in each of the 4 inter-electrode
regions at a distance of approximately 13m from the centre (7m in from the edge).
7
Also shown in this figure (9b) is the variation in the magnitude of E 2 in this plane.
Although the variation of E 2 in the z-direction is far greater than that which occurs
in the in the x-y plane, these vectors show that under conditions giving positive DEP,
the gradient of the DEP force is such as to move the particles inwards towards
discrete locations as observed experimentally. Particles move down the gradient of
DEP force to this point where both E 2 and the electric field is at a maximum and
where a net balance of convective and dielectrophoretic forces exists.
5. Conclusion
The dielectrophoretic behaviour of the mammalian virus HSV-1 in microelectrode
structures has been investigated over the frequency range 10kHz to 20MHz. With
particles suspended in an iso-osmotic EDTA/mannitol medium of conductivity
5mSm-1 both positive and negative dielectrophoretic collection was observed. The
transition between these two types of behaviour at a frequency in the range of 4 - 5
MHz is compatible with a simple physical model of the virus. The increasing positive
dielectrophoretic collection, which is observed at low frequencies, is considered to
arise from the polarisation of the diffuse double layer surrounding the virus. In
addition to the DEP force an electrohydrodynamic force causes small aggregates of
particles to form and levitate at points of electric field maxima.
Acknowledgements
The authors would like to thank Mrs. Joyce Mitchell and Ms Mary Robertson for
virus preparation, Mr. William Monaghan for electrode fabrication and Mr. Nicolas
Green for valuable discussions. This work is supported by the Biotechnology and
Biological Sciences Research Council (UK) grant no. 17/T05315.
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6. References
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Huang Y, Holzel R, Pethig R and Wang X-B (1992) Phys. Med. Biol. 37
1499-1517
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Markx GH, Huang Y, Zhou X-F and Pethig R (1994) Microbiology 140
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Green NG, Morgan H and Milner JJ (1997) J. Biochem Biophys. Methods 35
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Whitley RJ (1996) Fields’ Virology ed. Fields BN et al. (Lippincott-Raven)
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Huang Y and Pethig R (1991) J. Meas. Sci. Technology. 2 1142-1146
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Talary MS and Pethig R (1994) IEE Proc.- Sci. Meas. Technol. 141 395-399
9
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Biophys. Acta 1034 93-101
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Ramos A, Morgan H, Green NG and Castellanos A (1998) (Submitted to
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Melcher JR and Taylor GI (1969) Annual Review of Fluid Mechanics (Palo
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Figure Legends
Figure 1. (a) A transmission electron micrograph of a Herpes Simplex Virus, Type 1
(HSV-1) particle. (b) Schematic diagram of the HSV-1 virion showing the DNA,
Capsid, Tegument and Membrane.
Figure 2. A photograph of the electrode microstructures used in these experiments.
The area of the electrode corresponding to the electric field simulation of figure 8 is
shown in the box. Scale bar: 20m. The electrode is 20m from centre to edge and
has a gap in the centre of 6m.
11
Figure 3. A sequence of 3 fluorescence photographs showing the time course of
Herpes Simplex Virus, Type 1 particles collecting in the arms of a polynomial
electrode under positive dielectrophoresis. The photographs were taken 30 (a), 60 (b)
and 90 (c) seconds after application of a 500kHz, 5V peak to peak sinusoidal signal.
Images were taken with an image intensifying camera (Photonic Science ISIS II) and
recorded on S-VHS video.
Figure 4. A sequence of fluorescence photographs showing the time course of HSV1 particles experiencing negative dielectrophoresis and collecting in the centre of the
electrode. The photographs were taken at 30-second intervals (a,b,c) after application
of a 10MHz, 5V peak to peak sinusoidal signal. Aggregation of particles is achieved
rapidly and is limited in size by the profile of the potential energy well.
12
Figure 5. A sequence of fluorescence photographs showing the time course of HSV-1
particles collecting in the centre of the electrode 30, 60 and 90 seconds (a,b,c) after
application of a 100kHz, 10V peak to peak sinusoidal signal. A large aggregate is
visible at the centre of the trap, which can attain a radius in excess of 15m.
Figure 6. A fluorescence photograph showing the aggregation of particles both at the
centre of the trap, and at stable points above the inter-electrode regions, 2/3 of the
distance between the centre and edge of the trap. The particles were collected using a
5V peak to peak, 30kHz applied sinusoidal signal.
13
Figure 7. A graph of the rate of collection of virus particles in the electrode trap as a
function of applied field frequency.
The rate was determined by measuring the
increase in fluorescence across the electrode array at 30-second intervals after
applying the field.
14
Figure 8. A Clausius Mossotti plot, equation (2), showing the variation in the sign and
magnitude of the force on a virion as a function of frequency. Values used in the
simulation were: membrane conductivity < 10-8Sm-1, surface conductance, Ks =
-1
3.5nS, membrane permittivity, m = 10, tegument conductivity, i = 5mSm ,
tegument permittivity, i = 70. The medium conductivity used for this plot is med =
5mSm-1. This figure indicates that the model is consistent with the experimental data
above a frequency of 250kHz, but does not predict the increase in the magnitude of
the positive DEP force observed at low frequencies in the collection rate data.
15
Figure 9. A plot of the spatial variation of the normalised RMS electric field in a
plane 10m above the electrode surfaces, in the region defined by the box in figure 2
is shown in (a). The electric field is at a maximum in between the electrode arms,
approximately 13m from the centre of the electrode array. Also shown (b) is the
variation in the DEP force across the same plane. The vectors point to the maximum
in electric field as seen in figure 9(a).
16