Microfluidic Device for Simultaneous PulsedElectric Field Electroporation and Dielectrophoresis

Studies of Single Biological Cells1Elham Salimi, 1Marija Nikolic-Jaric, 1Tim Cabel, 2Katrin Braasch,

2Vincent Jung, 2Michael Butler, 1Douglas J. Thomson, 1Greg E. Bridges1Department of Electrical and Computer Engineering

2Department of MicrobiologyUniversity of Manitoba

Winnipeg, Manitoba, CanadaEmail: umsalimi@cc.umanitoba.ca, Greg.Bridges@ad.umanitoba.ca

Abstract—We present a microfluidic device able to electropo-rate and measure the resultant changes in dielectric properties ofsingle biological cells. The device can measure the dielectrophore-sis (DEP) response of the electroporated cell from a few secondsafter exposure to electroporating pulses. Studying electroporationat the single cell level can provide insight on the electroporationprocess as well as a method of differentiating cellular state (e.g.normal and cancerous). Moreover, combining electroporation anddielectric characterization into a single device eliminates the needfor post-sample analysis as required in techniques such as fluo-rescence cytometry. Experiments performed on Chinese hamsterovary (CHO) cells demonstrate the ability of the device to detectchanges in the DEP response of individual cells immediately afterbeing exposed to microsecond duration electroporating pulses.

I. INTRODUCTION

Increasing the permeability of a biological cell membraneusing intense pulsed electric fields has found numerous biolog-ical and medical applications such as electrogenetherapy, elec-trochemotherapy, and irreversible electroporation of cancerouscells [1]–[3]. The phenomenon that is called electroporationfacilitates the cell uptake of impermeable materials by makinghydrophilic pathways in the cell membrane through which ionsand polar molecules can pass and enter the cytoplasm. Elec-troporation affects the dielectric properties of a cell membraneas the created pores are significantly more conductive than theintact parts of the membrane [4]. This occurs on a very shorttime-scale. On larger time-scales, the cell internal propertiesare altered due to ion transfer across the membrane. Hence,monitoring variations of dielectric properties of a cell afterelectroporation can be employed as a detection technique toexplore a cell’s interaction with an external electric field and itssubsequent physiological change. The dielectric spectroscopytechnique has been used to monitor changes in the dielectricproperties of a population of electroporated cells (bulk mea-surement) [5].

Measuring the DEP response of cells - migration of cellsin a non-uniform electric field towards or away from regionsof high field intensity - is a technique commonly used tostudy their dielectric properties. DEP has been widely used

to separate various types of cells based on differences in theirdielectric properties and geometry [6]–[8]. In this study a DEPcytometer is employed to monitor changes in the dielectricproperties of individual cells immediately after electropora-tion. This provides valuable information on the cell ion fluxafter electroporation and how the external electric field affectsthe cell membrane. Moreover, the effects of different pulseparameters including the pulse duration, amplitude, rise-time,total number of pulses, and the delay time between multiplepulses applied to individual cells can be explored to optimizethe electroporating pulse parameters for a specific applicationand type or state of a cell.

In this paper we present a microfluidic system capableof detecting and electroporating single biological cells. Thesystem measures the DEP response of single cells immediatelybefore and after electroporation and can do this over anextended period of time. We use the system to study gradualchanges in the dielectric properties of CHO cells.

II. MATERIALS AND METHODS

A. Electroporation

The physical process of electroporation involves formationof hydrophilic pores in the membrane of cells. Since thecreated pores are highly conductive their formation can beelectrically modeled as an increase in the membrane con-ductivity. Simulations of microsecond electroporation of cellspredict approximately two orders of magnitude increase inthe cell membrane conductivity at electroporated areas [4].With reversible electroporation 30 % of the cell membranecan become electroporated [9] and the average increase in themembrane conductivity becomes

𝜎𝑚(𝑎𝑣) = 0.7𝜎𝑚0 + 0.3𝜎𝑚𝑒𝑝. (1)

where 𝜎𝑚0 and 𝜎𝑚𝑒𝑝 are the conductivity of the membrane atintact and porated areas, respectively.

The opened pores also allow the transfer of ions into andout of the cell [10]. This causes changes in the cytoplasmconductivity and consequently the cell polarizability.

Fig. 1: (a) 𝑅𝑒 {𝐾𝐶𝑀} vs. frequency for a typical viable non-porated CHO cell in a medium of conductivity 0.17 S/m.

B. Cells Response to Dielectrophoresis Forces

When a cell is subjected to a non-uniform electric field aforce, dielectrophoresis (DEP), is exerted on it that is directedalong the gradient of the electric field and is expressed as [11]

𝐹𝐷𝐸𝑃 =3

2𝜖𝑒𝑉𝑐𝑒𝑙𝑙𝑅𝑒 {𝐾𝐶𝑀} ∇⃗(𝐸2

𝑟𝑚𝑠) (2)

where 𝜖𝑒 is the permittivity of the medium, 𝑉𝑐𝑒𝑙𝑙 = 43𝜋𝑅

3

is the volume of the cell, and 𝐸𝑟𝑚𝑠 is the rms value of theelectric field at the center of the cell. 𝑅𝑒 {𝐾𝐶𝑀} is the realpart of the Claussius-Mossotti factor which is a measure ofthe cell’s polarizability in the medium and is given by

𝐾𝐶𝑀 =𝜖𝑐 − 𝜖𝑒𝜖𝑐 + 2𝜖𝑒

. (3)

In (3) 𝜖𝑒 and 𝜖𝑐 are the complex permittivity of the media andthe cell defined as 𝜖 = 𝜖 − 𝑗 𝜎

𝜔 with 𝜔 being the frequencyof the electric field. 𝜖𝑐 is an effective value incorporatingthe electrical properties of the complex internal structure.Depending on the sign of 𝑅𝑒 {𝐾𝐶𝑀} the DEP force canbe with or against the gradient of the electric field - termedpositive or negative DEP, respectively (Fig. 1). The Claussius-Mossotti factor is dominated by the conductivity of the cell andthe medium at low frequencies whereas at high frequenciesit is mostly affected by their permittivities [12]. Given thatelectroporation causes remarkable change in the conductivityof the cell plasma membrane we hypothesize that studying theDEP response of a cell at low frequencies (100 kHz-10 MHz)can provide valuable information on the effect of applyingelectroporating pulses.

III. EXPERIMENTAL SETUP

A microfluidic device with coplanar electrodes at the bottomof the channel along with an RF interferometer is used to bothelectroporate single biological cells and monitor the changesin their DEP response after electroporation. The microfluidicdevice and the electrode configuration are shown in Fig. 2.A dilute sample of cells is introduced to left and right inlets

Fig. 2: (a) The microfluidic device and (b) the incorporatedelectrodes designed for detection, electroporation, and DEPstudy of single biological cells.

of H channel, ports I1 and I3 (Fig. 2.a). The velocity of theflow in the microfluidic channel as well as the direction ofthe flow can be set by changing the pressure applied to theports. The concentration of cells and the pressure of the portscan be controlled so that multiple passes of the cell acrossthe electrodes can be achieved (back and forth motion). Asa cell flows over the array it is detected and actuated bythe electrodes. The two side electrodes, labeled as S1 andS2 (Fig. 2.b), are sensing electrodes that are energized bya low amplitude (100 mV) gigahertz frequency signal. Theelectric field generated across the channel by these sensingelectrodes does not exert a significant DEP force on the cellsdue to its low intensity. The middle electrodes, labeled as A,are used to apply electroporating electric pulses to cells aswell as generate a dielectrophoresis actuating force on them.To electroporate a cell a number of microsecond durationpulses are applied to the cell as it passes over the actuatingelectrode. Immediately after electroporation the electrodes areenergized by a megahertz frequency sinusoidal voltage toactuate the cell by a DEP force. To reduce uncertainty in theDEP measurement, the cell is cycled back and forth over theelectrodes and the resulting DEP response is averaged over 20cycles. The detection and actuation techniques are discussedin more detail in the following sections.

A. Detection

A cell in the microfluidic channel is detected by sensingthe capacitance change, Δ𝐶, that the presence of the cellinduces between the sensing electrodes. As a cell flows overthe coplanar electrodes it displaces the media with the differentdielectric property of the cell which results in a change in thecapacitance between the electrodes. Details of the apparatusand the interferometric system that is used to detect Δ𝐶 isexplained in [13]. It can be shown that Δ𝐶 is

Δ𝐶 = 3𝜖𝑒𝑉𝑐𝑒𝑙𝑙𝑅𝑒 {𝐾𝐶𝑀} 𝐸2𝑟𝑚𝑠

𝑈2𝑟𝑚𝑠

(4)

where 𝜖𝑒 is the medium permittivity, 𝑉𝑐𝑒𝑙𝑙 is the volume of thecell, 𝐸𝑟𝑚𝑠 is the rms value of the electric field at the center of

S1S2

A

nDEP

pDEPno DEP

e [s]

NoDEP

Time

Sign

al a

mpl

itude

P1

P1

P1P2 P2P2

P1>P2

P1=P2

285 um

100 um

40 u

m

P1<P2

Fig. 3: Example DEP induced signals (top) for a CHO cell asit passes over the sensing electrodes, S1 and S2 (bottom). Thesolid lines (bottom) show typical flow trajectories.

the cell, 𝑈𝑟𝑚𝑠 is the rms value of the electric voltage appliedto the electrodes, and 𝑅𝑒 {𝐾𝐶𝑀} is given by (3). As expectedthe measured Δ𝐶 is a function of the dimensions and electricalproperties of the cell as well as the amplitude of the electricfield.

The gigahertz sensing voltage is applied to the sensingcoplanar electrodes, and is concentrated between the electrodesS1 and S2. The signal as a cell passes over the electrodes,referred to as the cell signature, has its maxima as the cellpasses between the electrodes S1 or S2. Fig. 3 (bottom) showstypical signatures for a CHO cell.

B. Actuation

For the coplanar sensing electrode pairs the intensity ofthe electric field decreases rapidly as the distance from theelectrodes increases. Therefore, the capacitance change sensedby these electrodes and consequently the amplitude of asignature varies with altitude in the microfluidic channel (asformulated in (4)). Based on this a differential measurementcan be performed to measure the DEP force that acts on a cell.As a cell enters the electrode region (from left to right) thefirst set of sensing electrodes registers the peak marked as 𝑃1

in Fig. 3. As the cell moves over the actuating electrodes it isexposed to a DEP force that causes the cell to move towardsor away from the electrodes. As the cell reaches the second setof sensing electrodes the second peak, 𝑃2, is registered. Theamplitude of 𝑃2 can be greater or smaller than 𝑃1 dependingon the direction of the DEP force. 𝑃2 > 𝑃1 when the force isattractive (pDEP) and 𝑃2 < 𝑃1 when it is repulsive (nDEP).The recorded signatures of a CHO cell actuated by nDEP andpDEP forces are shown in Fig 3 (middle and right). Forceindex parameter, 𝜙, quantifies the change in the signature dueto DEP actuation as,

𝜙 =𝑃2 − 𝑃1

𝑃2 + 𝑃1. (5)

Here 𝜙 is a direct measure of the vertical translation of thecell due to the DEP force. As discussed in the previous sectionthe DEP force is related to the cell volume, real part of theClaussius-Mossotti factor, and the gradient of the electric fieldat the center of the cell (as formulated in (2)). Thus, for asingle cell moving in the microfluidic channel changes in theDEP force is attributed exclusively to changes in the Claussius-Mossotti factor and the dielectric properties of the cell.

In order to measure the gradual changes in the dielectricproperties of a single cell as the result of electroporationwe monitor variations of 𝜙 over a period of time startingimmediately after exposing the cell to electroporating pulses.

IV. RESULTS

In order to verify the ability of the device to electroporatecells and measure their DEP response experiments are per-formed on CHO cells suspended in a medium of conductivity𝜎𝑚 = 0.17 𝑆/𝑚. The cell solution is adapted from [14]with isotonic osmotic pressure as the CHO growth media.The procedure used is as follows. The DEP response of anuntreated cell is first measured by applying a 6 MHz, 8𝑉𝑝𝑝 DEP voltage to the actuating electrodes and moving thecell back and forth over the sensing and actuating electrodesseveral times and recording the force index. This is done bychanging the direction of the flow to move the cell back andforth in the channel. Then the cell is exposed to one or multipleelectroporating pulses with amplitude of 3 kV/cm, duration of100 𝜇sec, and rise/fall-time of 100 nsec. The DEP responseof the cell is measured periodically after electroporation for30 minutes. The gradual change of the force index (related tothe cell DEP response by (5)) over 30 minutes after multiplepulse applications is shown in Fig. 4. In this example, the cellis subjected to two separate electroporating applications withan interval of 10 minutes between them. In each applicationtwo pulses of 3 kV/cm intensity, and 100 𝜇sec duration areapplied to the cell with repetition frequency of 20 Hz. Controlmeasurements with no DEP actuation were performed aftereach group of 20 DEP actuated signatures to verify that the cellsignature is symmetric, 𝜙 = 0, when there is no DEP actuation.The plot shows decrease in the value of 𝜙 approximately 1minute after the application of first pulses. After the secondexposure the force index drops to zero and goes negativefor the next 15 minutes reflecting dramatic changes in thedielectric properties of the cell. The result demonstrates theability of the system to monitor induced changes in thedielectric properties of a cell due to electroporation a fewminutes after exposure.

V. CONCLUSION

A label free technique based on the DEP response of cellswas used to study the electroporation of single biologicalcells. The technique was implemented in a microfluidic device.Experiments performed on CHO cells demonstrated the abilityto detect changes in the DEP response of individual cellsimmediately after being exposed to microsecond durationelectroporating pulses. The device can be employed to study

Fig. 4: Force index vs. time for a cell subjected to two separateelectroporating applications with an interval of 10 minutesbetween exposures.

the electroporation process of different cells or study changesand time-constants related to ion flux due to electroporation.

ACKNOWLEDGMENT

This work was supported by the Natural Sciences andEngineering Research Council of Canada, Western EconomicDiversification Canada, and CMC Microsystems.

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