Manipulation of Herpes Simplex Virus Type 1 by Dielectrophoresis.
Michael P. Hughes, Hywel Morgan[*] , Frazer J. Rixon, Julian P. H. Burt3 and Ronald Pethig3
Bioelectronics Research Centre
University of Glasgow
Glasgow G12 8QQ
Scotland, UK
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.
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.
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:
(1)
where is the absolute permittivity of the suspending medium, E is the local (rms) electric field, Ñ is the del vector operator and is the real part of the Clausius-Mossotti factor, defined as:
(2)
In equation (2) and are the complex permittivities of the particle and medium respectively, where and e is the permittivity, s the conductivity, w the angular frequency of the applied field, and j = . Depending on the relative magnitudes of and , 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 ÑE 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 across gaps of 2mm or less. This has allowed researchers to dielectrophoretically manipulate sub-micron 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 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 HSV-1 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 10-20nm 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 2mm between nearest neighbour electrodes and 6mm 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 50mm and a sample volume of the order of 1ml. 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. 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 7mm above the electrodes in a column approximately 2mm diameter and 50mm 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 mm diameter field funnel formed by the electrodes, the spherical aggregation of virions grew to a radius of 20 mm 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 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 as well as a surface component. The total conductance of the membrane can be written [20,21] as: where is the surface 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 diameter of HSV (250nm) the surface conductance dominates, i.e. (for < 5 x 10-3 Sm-1). As shown in figure 8, a good fit to the experimental data occurs 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. 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.