Proceedings of the IEEE International Conference on Mechatronics & Automation Niagara Falls, Canada • July 2005

0-7803-9044-X/05/$20.00 © 2005 IEEE

Carbon Nanotube Enhanced Pulsed Electric Field

Electroporation for Biomedical Applications J. D.Yantzi & John T.W. Yeow*

Department of Systems Design Engineering, University of Waterloo, Waterloo, ON, Canada.

*corresponding author: jyeow@engmail.uwaterloo.ca

AbstractPulsed Electric Fields (PEF) are commonly used in

microbial electroporation applications. Emerging lab-on-

a-chip technologies use PEF to control membrane

permeability to access internal cellular contents including

nucleic acids and proteins. If micropores in the membrane

(created by E field) are significant in terms of size,

persistence time and number, in comparison to the overall

membrane area, the conditions for mechanical destruction

of the cell membrane are favoured. Membrane

breakdown is associated with cell death and dispersal of

cell contents including nucleic acids and protein. This

paper serves as an initial investigation into the use of

carbon nanotubes (CNT) to reduce the voltage

requirements for irreversible electroporation for portable

lab-on-a-chip devices with strict power limitations.

Index Terms: Electroporation, Cell Lysis, Carbon

Nanotubes, Lab-on-a-Chip

I. INTRODUCTION

Lab-on-a-chip (LOC) is a concept that adapts commonly used

laboratory procedures into automated processes, carried out on

platforms small enough to be contained within a portable,

hand-held device. This technology was brought to conception

through the advent of standardized IC technology, enabling

the fabrication of patterned microstructures such as electrodes

and fluidic channels on “chips” amenable to control

applications. Some of the advantages LOC systems have over

conventional “macro” scale labs include reduction in

reagents/waste, faster processing times, elimination of bulky

sample handling steps, complete automation and portability. A

successful lab-on-a-chip application requires multiple

functions integrated to complete a complex task. For example,

to create a LOC device capable of carrying out diagnostic

genetic tests on blood samples several modules must be

integrated; 1) accessing DNA within the cell, 2) separation

and transport of various cellular fractions and 3) detection of

specific DNA sequences. In fact, micro-fabricated devices

devoted to carrying out nucleic acid purification and analysis

protocols have already been realised [1],[2],[7]. The same

processes in a traditional lab would be carried out using

chemical reagents, centrifugation steps and a trained

technician- antiquated processing steps that can be replaced by

small foot-print devices that are inexpensively mass-produced

through industry standardized fabrication processes.

The research presented here investigates a novel method of

addressing the first step in biotech diagnostic applications,

namely biomolecular access. The extraordinary electrical

properties, and extremely small dimensions of carbon

nanotubes have been used to enhance the capabilities of pulsed

electric fields (PEF) as a method of releasing the biomolecular

contents of the cell for diagnostic lab-on-a-chip applications.

II. THEORY

A. Pulsed Electric Field Cell Lysis Common methods for cell lysis in macro scale processes

include sonication, bead milling and chemical treatment.

However, application of pulsed electric fields to cells in

suspension has become a popular method to access cellular

contents for lab-on-a-chip applications. Chemical methods

require additional contaminating reagents which need to be

subsequently removed to avoid interference with downstream

processing steps. Sonication can lead to the formation of toxic

compounds, while bead milling is difficult to miniaturize and

can generate excessive heat. PEF methods, on the other hand,

require no additional reagents and are produced using

structures that are easily achieved through common

lithographic techniques. High voltage ac or dc pulsed electric

fields are used to generate increased charges across the

membrane leading to cell lysis. A normal dc field could

achieve the same effect, but will cause electrolysis and ohmic

heating due to excessive energy input which is avoided by

pulsing the field [3]. Electrolysis generates bubbles of gasses

which can interfere with microfluidic handling, produces

unwanted electrolytic chemical compounds and leads to

degradation of the electrode materials. Ohmic heating can

have adverse effects on thermo-sensitive compounds like

proteins and potentially adverse effects on reaction kinetics of

a given application. By varying the duty cycle and/or applying

very short pulses the energy input is greatly reduced,

preventing the previously mentioned effects while still

achieving the increased trans-membrane potential causing

electroporation.

A cell exposed to an electric field gradient experiences a

build-up of apposing charges across its membrane. A typical

resting potential across a cellular membrane is 10 mV and

causes the build-up of ions across the membrane[5]. If the

transmembrane voltage is increased, the force of the ions

across the membrane builds to the point where the thickness of

the membrane is reduced. If the field is intense enough, pores

form across the membrane allowing an open path between the

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cellular matrix and the external suspending media. Depending

on the magnitude, duration and frequency of the applied

pulse, the membrane can either reseal (reversible

electroporation) or remain open (irreversible electroporation).

Irreversible electroporation results in a failure of the cell

membrane to maintain essential ionic balances across the

membrane and the cell ruptures (Fig.1).

Fig. 1. Effects of Electric Field on Membrane Integrity, where ECM and ICM

are internal and external cell medium respectively. a) Normal ionic balance

across cell membrane, b) compression of membrane due to increased

attraction of ions caused by increased electric field, c) membrane unable to

resist compression and a pore in the membrane is formed.

Both reversible and irreversible electroporation have

applications in biotechnology and medical research.

Reversible electroporation has been used for years as a method

of introducing DNA and proteins into cells [6]. Biological

cells are slightly negative in charge and as a result, it is

difficult to introduce other polar molecules like DNA and

proteins. Using reversible electroporation methods, pores in

the membrane are created long enough to allow DNA, proteins

or drugs to slip into the cell before the pores reseal and the cell

remains viable. The food Industry has used irreversible

electroporation to sterilize water, juices, eggs and soup

without heating which can result in off tastes and degradation

of ingredients [5]. However sterilization isn’t the only

application of irreversible electroporation or cell lysis. Several

lab-on-a-chip type applications have been produced that use

this technology to liberate the biomolecular contents of the

cell for diagnostic purposes [14], [15],[16],[18]. Cheng et al

created a device capable of collecting bacteria from a

background of blood cells followed by subsequent lysis, to

access nucleic acids for analysis [7]. Typically a voltage of 1V

across the cell membrane is required to rupture the cell

membrane [8], however the field strength required depends

greatly on cell type/shape, media conductivity and media

temperature [5]. The critical field required for cell lysis can be

calculated by:

faV

E cc (1)

where Vc is the voltage, a is the radius of the cell and f is a

geometric form factor which is dependent on cell type [13].

The pulse duration (or charging time constant, ) required to

reach the critical voltage across the membrane of a spherical

cell is expressed by:

Cr2

1

2 (2)

Where 1/ 2 are the resistivities of the suspending medium and

cytoplasm respectively, C is the capacitance of the

membrane/unit area and r is the radius of the cell [13].

Using micro-fabrication technologies provides smaller gaps

between electrode structures thereby enhancing field intensity

capabilities. Reducing power requirements is a key demand in

portable device operation, so devices must be designed to

minimize their energy needs. Irreversible electroporation or

cell lysis is cited to occur at around 4 kV/mm [4], although

others studies indicate upwards of 10 kV/mm [9]. While in

[17] a critical field strength as low as 1.4 kV/mm was cited as

the critical field strength for lysis of E. coli. Experimental

setup and varying media conditions can no doubt account for a

significant portion of this discrepancy. The aim of this paper is

to lower the voltage requirement of electroporation through

the use of nano-structured materials. Thereby reducing the

voltage requirements, so that processes like PEF cell lysis

which require kilovolts to operate on the macro scale can be

achieved using a battery operated hand-held device.

B. CNTs & Supercapacitance Carbon nanotubes (CNT) were discovered by Iijima in 1991

[10]. Through application of high voltages, graphite sheets can

be rolled seamlessly onto themselves forming tube-like

structures with widths of less than a nanometer and many

microns in length. CNT have remarkable mechanical strength,

are chemically inert, and behave as conductive metals. These

properties make this material for applications in nano-

electronic circuits, hydrogen storage for fuel cell technology

and supercapacitors capable of delivering increased energy

outputs. Capacitors store energy in the form of separated

electrical charge. Covering the electrode of a capacitor with

conductive nano-structured materials has improved the

performance capabilities of electrochemical energy storage

systems [11]. This development has lead to a new breed of

batteries with longer lifetimes and greater output capabilities.

CNTs enhance traditional electrode configurations via their

+ +

- - -

+ + + +

+ + + +

- - - -

- - - - -

E

E

E

a)

b)

c)

Pore in Membrane

ECM

ICM

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low resistivity, prolate dimensions and increased surface area

characteristics [12]. Instead of using flat plates to achieve

capacitance, CNTs can be patterned on the electrodes resulting

in higher electric fields and power output at lower voltages.

Fig. 2 SEM image of CNT used in this study to enhance electric

field properties

In addition to lowering the voltage requirements for PEF cell

lysis, the concentrated field regions produced by the CNT

represent an effective means of disrupting smaller sub cellular

organelles, viruses and membrane bound nuclei present in

eukaryotic cells. As previously stated, typical transmembrane

potentials for membrane breakdown are close to 1 volt. In fact,

a recent study also made use of the unique properties of

carbon nanotubes for electroporation [12]. Rojas-Chapana et

al, used the “lightening rod effect” of CNTs mixed within cell

suspensions to create localized high field regions at the tips,

creating pores in the membrane, permitting the uptake of

extra-cellular substances. An expression of the degree to

which the electric field is enhanced at the tips of carbon

nanotubes is given by:

DL

EE

o

(3)

Where is a constant, L is the length of the CNT and D is the

diameter [12]. A closer look at the L/D relationship

demonstrates why long thin structures are so effective at

concentrating field.

The results of Chapana et al also indicated that there was a

tendency of the carbon nanotubes to collect and adhere to

lipid membrane structures. When microwave pulses were

applied, the combined effect was used to transport substances

into cells. A technique commonly used in tissue culturing to

introduce DNA or special compounds for research and

pharmacological applications.

III. EXPERIMENTAL

The CNT enhanced PEF cell lysis device consists of three

layers which comprise the electroporation chamber; an

aluminium plate with 1cm × 1cm patch deposited with

randomly aligned single walled carbon nanotubes from

Xintech Inc. A patterned, 50 μm thick shim layer provided the

microchannel structure which enclosed the CNT patch as well

as fluidic inlet and outlet ports on the aluminium plate. The

ceiling of the microchannel comprised of a glass substrate

with patterned electrodes made up copper tape (3M). The

three layers were clamped together to prevent leakage and E.

coli samples suspended in deionized water were passed

through the system with a micro-peristaltic pump with

controllable flow rates (Instech Model P720). Cell samples

were periodically mixed during the experiment to prevent any

settling phenomena that could result in cell concentration

fluctuations. The powering electrode was connected to a solid

state relay (VS Holdings LCC, model 7107D3F500D6) circuit

that controlled the duty cycle and frequency provided by a

DAC card (National Instruments PCI 6711). The applied

frequency used in the results section was held at 10 kHz for all

runs. The control voltage of the signal from the circuit was

supplied by a 100V power supply and was stepped up by an

EMCO K Series device for the setup allowing for voltages in

excess of 100V. The floor plate of the device was grounded,

providing an electric field through the 50 μm thick

microchamber. Two separate setups were prepared to compare

the relative effect of a regular conductive aluminium floor

plate vs. an aluminium plate with deposited CNTs. All other

setup parameters were held constant.

Fig 3 Experimental setup showing cell sample, pump, electroporation

device and relay circuit.

Analysis was performed using DH5 E. coli cells equipped

with a kanamycin antibiotic resistance gene. Cells were

Relay

Circuit

Lysis Device

Pump

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prepared by incubation overnight in a 37˚C shaker in LB

nutrient broth. 2 ml aliquots were centrifuged at 3000 rpm for

3 minutes and the resulting pellet was washed with deionized

water and resuspended in 20 ml deionized water. E. coli cells

suspended in deionized were passed through each setup at a

continuous flow rate of 10 μl/second. The duty cycle used for

data presented in results section was 50%. Samples were

exposed to electric fields covering a range of voltages as they

passed through the device. 500 μl samples were collected from

a fluidic outlet port for each applied voltage and thoroughly

mixed before plating 20 μl on LB agar plates containing the

antibiotic kanamycin using fully sterile techniques. The plates

were then incubated for 16 hours at 37˚C and the number of

colony forming units (CFU) was totalled. 0 volt positive

controls were included in each experimental run to

demonstrate samples with 0% cell lysis. The number of CFU

for each voltage sample was compared against the positive

control to give a relative indication of the % non-viable or

lysed cells, and results were averaged over several runs. As a

means of verifying cell lysis, aliquots of plated E. coli which

showed almost 0% viability (determined by colony counting)

were imaged using phase contrast microscopy using a 100X

oil immersion lens with a Leica DMRA2 microscope and

compared to the 0 volt positive control (fig. 7 ).

IV. RESULTS & DISCUSSION

The voltages required for >95% reduction in E. coli cultures

was greatly reduced using electrodes covered with CNT. With

the setup used in this research upwards of 135 volts were

required to achieve the dramatic reduction in percentage of

viable cells. However, with the setup including CNT the

voltage was reduced to around 35V. Although the 4-fold

reduction in voltage is very significant, this factor could no

doubt be further reduced by altering various setup parameters

including shallower channel heights and covering both

electrodes of the capacitor setup with CNT. In the current

setup the electric field is most intense around the floor of the

channel, however cells that pass through the device at higher

level will be exposed to less intense fields and could affect

resulting colony counts. Fig 4 shows typical colony counts

using the device without carbon nanotubes. The positive

control a) has a lawn of bacteria while there is a marked

reduction in colony numbers as the voltage is increased b)-d).

Fig. 6 shows the voltage requirements of both the device fitted

with carbon nanotubes and the device without. The same type

of reduction in colony forming units is demonstrated in both

curves, however, the voltages required are significantly

reduced for the setup employing CNT field enhancing effects.

These experiments were repeated many times and the values

were averaged and plotted (fig. 6). Phase contrast microscopy

was used to verify cell lysis (fig. 7) The results clearly show a

stark discrepancy between the two samples. Positive control

samples showed an abundance of in tact E. coli cells, while the

lysed samples had very few in tact cells and were replaced by

what seemed to be cell debris and fragments of the membrane.

Fig. 4 Relative colony counts after exposure to different voltages;

a) 0 volt control sample b) 50 V c) 100 V d) 125 V

Fig 5 Relative colony forming units (CFU) using CNT enhanced

PEF a) 0 volt control >200 colonies, b) 8 volt sample >100

colonies c) 25 volt > 50 d) 35 no colonies.

In addition to lowering the overall voltage requirements, the

concentrated field around the carbon nanotubes tips could be

used for applications where the size of the cell or compartment

is very small. From (3) it is clear that the radius of the cell

plays a central role in the critical voltage for breakdown.

Therefore viruses or sub-cellular organelles which typically

have dimensions well under a micron can be much tougher to

lyse. The use of conductive nano-structured materials could be

used to provide increased field so that smaller biological

compartments can be ruptured with greater ease.

a) lawn of bacteria

c) <100 CFU d) <20 CFU

a) >200 CFU b) <100 CFU

c) > 50 CFU d) 0 CFU

b) >500

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Fig. 6 Graph of the effects of voltage on % cell lysis with and

without CNT

Fig. 7 Phase contrast images of positive control samples (a) and

samples exposed to high voltage pulses (b).

V. CONCLUSION & FUTURE WORK

This paper has presented the use of carbon nanotube

enhanced electric fields as an effective means of lowering

voltage requirements of irreversible electroporation.

Successful lab-on-a-chip systems will employ components

with low enough voltage requirements to run off a battery and

the contributions of this paper are a step in that direction. The

unique properties of CNTs however, could be extended

beyond their applications in irreversible electroporation and

future experiments will address this fact.

Acknowledgements

This research was made possible by the laboratory equipment

and biological samples provided by Dr. Perry Chou of the

Chemical Engineering department of the University of

Waterloo.

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