BIOTECHNOLOGY TECHNIQUES Volume IO No.4 (April 1996) p.221-226 Received 1st February.

HIGH FREQUENCY ELECTRIC FIELDS FOR TRAPPING OF VIRUSES

Torsten Mtiller, Stefan Fiedler, Thomas Schnelle, Kai Ludwig, Hartmut Junga and Gtinter Fuhr*

Humboldt-Universitat zu Berlin, Institut fXir Biologie, Invalidenstr. 42, 10115 Berlin, Lehrstuhl fti Membranphysiologie, (e-mail: Fuhr@rz.hu-berlin.de)

a Logware GmbH, Schwedenstrahe 9, 13359 Berlin

SUMMARY

Combining dielectrophoretic and hydrodynamic forces in micro electrode structures allows enrichment and stable trapping of viruses in aqueous solutions. Fluorescently labelled Influenza and Sendai viruses were collected from solutions of 2* 1 O5 - 2* 10’ viruses/y1 within a few seconds. In the central part of the trap a virus aggregate of about 2-9 urn in diameter was formed. This corresponds to a local enrichment of viruses up to a factor of about 1400.

INTRODUCTION

Under appropriate conditions, alternating (a.c.) electric fields give dielectric polarisation

forces which repel particles of lower effective permittivity than the surrounding medium from

electrodes (Pohl, 1978). Planar, quadrupole microelectrode-arrangements combine these

forces with sedimentation and hydrodynamic forces to entrap suspended particles in “field

funnels” or traps. Three-dimensional, octupole electrode-arrangements allow the creation of

electric field cages, which entrap objects by polarisation forces only. For suspended

submicron particles (such as viruses), Brownian motion becomes significant. Polarisation

forces scale with the third power of the particle radius whereas thermal motions vary inversely

with it. If these were the only considerations, particles with radii less than 500 nm could not

be trapped in water filled field cages (Pohl, 1978). However, miniaturised electrode-

assemblies allow the application of larger and more inhomogeneous electric fields (due to

increase in breakdown strength of aqueous solutions), both increase the polarisation forces.

Recently, it was shown that latex beads up to a diameter of 14 nm can be concentrated (Miiller

et al., 1995). This should also opens up new perspectives for handling of bacteria, viruses and

macromolecules.

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M A T E R I A L S A N D M E T H O D S

Microstructures: We have used electrode assemblies produced on glass wafers by optical lithography with dimensions typically between 5 and 25 ~tm (Fuhr et al., 1995). The planar electrodes were wetted with a 10 ~tl droplet of particle suspension and covered by a glass plate approximately 20 ~tm above the electrode surface. Closed field cages require a three- dimensional arrangement of electrodes. To achieve this, two planar electrode structures were mounted face to face, several micrometers apart (Fig. 1). The space between them is filled with a virus suspension, either directly or by structured micro-channel systems. The devices can be cleaned in an ultrasonic bath, cold or hot sterilised, and rinsed with alcohol or other liquids. Field generation: a.c. square wave pulses were applied using a Hewlett Packard generator HP-8116A. The frequencies were between 100 kHz and 50 MHz and amplitudes between 1 and 28 Vptp.

Viruses: Influenza virus (strain A/Japan and PR 8/34) and Sendai (strain Z) ( lmg protein/ml) were fluorescently labelled with Octadecylrhodamine B chloride (Molecular Probes, USA) of 10 ~tM at room temperature for 30 min in the dark, centrifuged, washed and resuspended in ice-cold phosphate-buffered 150 mM NaC1, then transferred to phosphatic-buffered 300 mOsm sorbitol using a Sephadex G-75 column. The final conductivity was about 74 mS/m and the final concentration was 0.2-1 mg protein/ml (1 mg protein/ml - 2.5 101l viruses/ml). Fusion activity of labelled viruses was proved by fluorescence dequenching assay (Hoekstra et a1.,1984). The virus behaviour and the evolution of the fluorescence signal was recorded by a microscope-video system (Leica Metallux 3, LD 50 objective with a CCD Micro Camera CS 3130 in shutter mode or with an Confocal Laser Scanning Microscope, Leica). The video processing was done with the software of the CLSM and a video printer. The whole experimental set up is shown in Fig. 1.

Epifluorescencec Microscope, caisra and uideo-sgstem

Sample droplet of uirus suspension

~ ~ I~LS)/ oc~opozs ~ field

cag~

H F - G e n e r a t o r 1 k H z - 50MHz

S i g n a l s , used f o r r o t a t i n 9 f i e l d t r a p

m_n_n_n_

@ d-Ln_r -LS

S i g n a l s , used for p l a n a r q u a d r u p o l s t r a p s N i t h alternatin 9 fields

@ _n_n_n u @ JXJ-U-u-ur

Fig. 1: Experimental set up Shown are a photo of a structure (left), a schematic view of one of the four cages, marked by arrows (middle) and the driving conditions (right). Top and bottom electrode planes are separated by an channel forming spacer. Bar : 100 Mm.

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THEORETICAL

The application of spatially inhomogeneous, high frequency a.c. electric fields, E, on particles

suspended in a liquid with different dielectric properties (permittivity E and conductivity o)

gives polarisation forces which repel particles of lower effective permittivity from regions of

high field strength - thus allowing the creation of field traps and closed cages (Fuhr et al.,

1995). For a spherical particle of radius R, the time averaged dielectrophoretic force (DEP)

where we have introduced the complex permittivity r = E + i00 (CO- radian frequency of

the electric field). For our electrode geometries a numerical procedure was necessary to

calculate the electric field. In addition, particle-particle (dipole-dipole and hydrodynamic

coupling) interactions, as present in particle suspension, can favour particle aggregation and

hence trapping. For small particles in an aqueous medium, acceleration can be neglected and

the trajectory of a particle can be determined by numerical integration of a stochastic

(Langevin) equation (see e.g. Fuhr et. al,. 1995). Due to the strong heating in the

interelectrode gaps the liquid becomes dielectrically inhomogeneous. As a consequence, the

heated water between the electrode gaps tends to be replaced by cold from the outside since it

has a higher permittivity. This is analogous to the well known effect that a medium with

higher permittivity tends to displace that with the lower value from a plate capacitor in

electrostatics. Thus, liquid streaming is induced. The direction of the streaming is determined

by convection. Small particles can enter the central part of the trap with the streaming and

build a large aggregate there. The aggregate cannot overcome the dielectric force barrier since

the DEP scales with R3 and the hydrodynamic force only with R (Stokes).

RESULTS

We have used influenza and Sendai viruses, fluorescently marked and suspended in an

aqueous solution. Viruses can be trapped in a field funnel produced by a planar electrode

system with dimensions typically between 5 and 25 urn (Fig. 2). Trapping and concentration

by negative dielectrophoresis occur within a few seconds of applying frequencies between

0.2 - 4 MHz and 6 - 14 Vptp. This experimentally observed time regime is in accord with the

calculations. The typical size of a virus aggregate was 8 urn for a electrode gap of 25 pm.

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Fig. 2: Time course of enrichment of Sendai viruses in dielectrophoretic fjeld traps - simulation (a-c) and experiment (d-f); (a,d) t=O s; (b,e) t4 s; (CA) t=g s

7..

+.

(b) ‘... .

I

(c) ‘!’ I For calculation (a-c), a diameter of Sendai viruses of 100 mn, a specific conductivity of 0.8 mS/m and a relative permittivity of 3 were assumed. Calculations were carried out for an octupole cage (electrode distance 25 urn) with sinusoidal 3 MHz rotating electric drive at a voltage of 15 V. Experiments (d-f) were carried out in a quadrupole trap with square wave a.c.-driving of the electrodes (l-3 MHz, 1 IV, electrode distance 25 urn, conductivity of the virus solution was 74 mS/m.) Time course of trapping in quadrupole and octupole structures was found to be similar. Optical detection is easier in quadrupole traps.

In a planar electrode arrangement, the particle behaviour is more complex, and the virus

concentration is a result of electric, sedimentation and hydrodynamic forces. Heating the

solution between the electrode gaps leads to anisotropic fluid properties as the conductivity

and permittivity are temperature-dependent. Therefore, space charges are induced and moved

in the electric field. Together with convection, this causes liquid streaming. In the axially

symmetric electrode configurations used, four whirls stabilise the virus aggregation in the

field funnel.

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To demonstrate the enrichment of virus particles inside the trap by the streaming, we compare

the starting particle concentration with the final one in the field trap at different dilutions. For

all tested dilutions (l/l - l/2000), viruses could be trapped (e.g. l/10 Sendai virus dilutions in

Fig. 2). There, the aggregate size after a few tens of seconds is approximately a third of the

electrode gap, e.g. about 9 pm in our structure. This corresponds to roughly 500,000 virus

particles or a local enrichment factor of about 1400. That means, most of the trapped viruses

were brought into the trap by electrohydrodynamically induced liquid streaming and

convection resulting in depletion of viruses in the bulk solution. In field cages only the

specimens originally within the trap region are confined. However, the combination of

octupole cages or saddle-point structure built with ring electrodes (Schnelle et al., 1996)‘with

linear electrodes and flow systems (Mtiller et al., 1993; Ma&x et al., 1994) should also allow

efficient filtering (Fig, 4). Another way to concentrate virus specimens uses antibody coated

latex beads. They can be stably held in dielectrophoretic field cages against liquid streaming

up to a velocity of about 200 urn/s.

Enrichment of viruses in a high frequency electric field is determined by their dielectric

properties and geometry. It should therefore be possible to separate different virus species.

The virus specimens used in our experiments were, however, similar both in geometry and

dielectric characteristics. The separation of slightly different particles requires force

magnification. This can be achieved by exploiting the electronic properties of the chamber

(resonance phenomena, Gimsa et al., 1996). The typical resonance frequency of our structures

is about 180 MHz. Separation of particle types is comparatively easy at frequencies where one

kind shows negative and the other positive dielectrophoresis. For similar specimens this

happens only rarely. Hence, the forces are weak and have to be amplified. This can be done by

shifting the resonance frequency of the chamber with active elements to relevant frequency

windows (Fig. 3). This should open up new vistas for detection and diagnostics of viruses and

macromolecules.

Fig. 3: Influence of a resonance on the DEP force near a cross over frequency (from positive to negative DEP); I a,b DEP curves of two ob.jects without resonance; 2 a,b with resonance

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Fig. 4: Particle separator based on ring-microelectrodes

Shown are three, ring-electrode systems (a,b,c). The electrodes are at top and bottom of a central channel (2) with openings to further channels (above (l), below(3)). Fluid and particles can stream through the central region of each electrode thus entering another channel. Liquid streaming through such a cascade can be cleared of micron and sub-micron particles. The separation procedure is illustrated in the three ring systems: a) Trapping of particles; b) Focusing to the openings; c) Release of particles to the neighbouring channel. Note, that by superimposing additional forces (e.g. hydrodynamic ones resulting from different streaming velocities in the channels) different kinds of particle could be separated.

Acknowledgement: We would like to thank Dr. B. Wagner from Fraunhofer Institut ftir Siliziumtechnologie (ISiT, Berlin) for manufacturing of microstructures; Dr. J. Gimsa for fruitful discussion about the resonance phenomena and Dr. S.G. Shirley (Humboldt- Universitat zu Berlin) for critical reading of the manuscript.

REFERENCES

Fuhr, G., Schnelle, Th., Hagedorn, R., Shirley, S.G. (1995). J.CeZl. Eng. l/l, 47-57. Hoekstra, D., de Boer, T., Klappe, K., Wilschut, J. (1984). Biochemistry 23, 5675-5680. Markx, G.H., Huang, Y., Zhou, X.-F., Pethig, R. (1994). Microbiology 140, l-7 Mtiller, T., Arnold, W.M., Schnelle, Th., Hagedorn, R., Fuhr, G., Zimmermann, U. (1993). Electrophoresis 14, 764-772. Mtiller, T., Gerardino, A.M., Schnelle, Th., Shirley, S.G., Fuhr, G., De Gasperis, G., Leoni, R., Bordoni, F. (1995). I1 Nuovo Cimento 17D, 425-432. Pohl, H.A. (1978). Dielectrophoresis, Cambridge: Cambridge University Press. Schnelle, Th., Mtiller, T., Fiedler, S., Shirley, S.G., Ludwig, K., Hermann, A., Wagner, B., Zimmermann, U., Fuhr, G. (1996). Naturwiss. in press Gimsa, J., Mtiller, T., Schnelle, Th., Fuhr, G. (1996). Biophys. J. submitted

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