Electrical Breakdown and Electroporation:
A Review of Experiments, theory and Applications
Hans Coster
悉尼大学科研院
INTERNATIONAL
BIOMEDICAL AND
BIOELECTRONICS
WORKSHOP
sydney.edu.au/research_support
Electrical Breakdown in Cell Membranes
Pulsed current - mA
Measured using intracellular electrodes (in cells of Valonia utricularis)
From Coster & Zimmermann 1975
Points
measured
in random
order
Membrane potential - mV
The Plasma Membrane of cells
F
F
Direct Effect: Application
of deformation force
(stress) produces an emf.
Electrical breakdown occurs
when the field strength in the
membrane is ~108 V/m
Plasma membrane of cells
surface
proteins
intrinsic
proteins
Lipids in a
bimolecular matrix
Mechanisms for electrical instabilities in cell
membranes
Pore formation in the lipid bilayer
Pores can reach a critical size that causes
rupture and the critical size is voltage
dependent.
Electrostriction of membrane proteins
Molecular electric
The intense electric field in the dipoles
membrane
remain
leads to a catastrophic electrostriction
aligned after removal
of the electric field
Both mechanisms can lead to a “runaway”
breakdown phenomenon.
Electrical properties of lipid bilayers:
Energy of ion partitioning
The Born Energy arising from image forces on
the ions is:
2e2  1

z
1
WB 
 

8 oR   m  w 
For a K+ ion this comes to ~3 eV
An ion partitioning energy of 3eV yields a
membrane electrical conductance ~ 20 orders of
magnitude less than the experimental values!
Electrical conduction on lipid membranes
Conclusion:
There must be another mechanism for ion conduction in lipid
bilayer membranes.
Pore defects
Pore defects in Lipid Bilayers
r
d
p = energy per unit area of the curved surface
m = interfacial free energy of the bilayer
The net energy to form a pore “defect”
EP   2rd P  2r 2 m
Critical instability in lipid membranes
Energy cost
Energy of pore
Net
Energy
r=Rc
Savings in
interfacial energy
r
The critical
condition
occurs when
Ep
=0
r
Voltage dependent pores & critical radius
V=0
V>0
r
Vm
Total energy of pore
Rc
Pore radius, r
Lipd bilayers become very
unstable at membrane potentials
> ~50 mV.
Instabilities at electrical breakdown
Measurements using intracellular
electrodes show that instabilities only
occur when the membrane potential
exceeds a well defined value.
Electrical breakdown in this case
occurred at -385 mV
Experiments also show that long trains
of subthreshold pulses do not lead to
electrical instabilities; that is, it is not a
probabilistic phenomenon.
This is not expected from the voltage
dependent pore model
200 mS
Electrostriction in membrane proteins
d
1
Energy W  CV 2
2
1  o 2

V per unit area
2 d
Electrostriction & electrical breakdown
Pe   dW  1  2o V 2
dd 2 d
Electric field induced
compressive stress
x d
dx
d
Pm  Y 
 Y ln
x
do
x d o
Elastic restoring stress
Electrical Breakdown occurs when:
 Pe    Pm
d
d
1

2
2
 0.3679Yd

V   

C 
p 0


o
Electrostriction & electrical breakdown
Theory
The full lines are the predicted
curves based on electrostriction.
Pulsed current
3 mA
Experiment
Using the initial ohmic
resistance from these
experiments, the theory fits the
experimental data with no
adjustable parameters!
R0 = 540 
R0 = 3350 
R0 = 3030 
0
500
1000
Membrane potential [mV]
1500
Electrical breakdown & Electroporation
Electrical breakdown, effectively produces pores or
defects in the cell membrane on a nano-meter scale.
If the current pulses are long enough, the large current
densities in the pores lead to the growth of the pore size
as well as electro-osmotic effects causing cell swelling.
This is often referred to as Electroporation.
Electro-poration and Electro-lysis
Applications: Electro-poration
Transfecting DNA into organisms (genetic
engineering)
Loading of cells with drugs as a therapeutic
“trojan horse”
Sterilisation of fluids (electro-disinfection)
Electrical cell fusion: involves additional
considerations
Dielectric Structure of Cells
solution
s , s s
Skin layer or
cell membrane
m , s m ,
thickness d
R
Core material
or
cytoplasm
c , s c
The effective dielectric
constant of such a particle is
frequency dependent
Clark Maxwell did this as an exercise in his treatise on
electromagnetism.
Electric Field Patterns around cells
q
E
E
q
++
++ +
m
--- - -
++ + ++
m
- - ---
EE
E
High frequencies
f > 10 MHz
-
m
m
-
+
Low frequencies
f < 30 kHz
Intermediate frequencies
30 kHz < f < 10 MHz
+
s,ss
- - -+ - + +
m
- -
c,sc
- - +- - + +
m
- -
Particle Dielectrophoresis
s < p
Effect of
Dielectric
constant
s
p
Force
s > p
s
p
Force
Dielectrophoretic Force
F  Re[( m ) E ]
~ ~
m  
~
Re: Real part of the function
is the induced dipole moment
~
For a cell surrounded by its plasma membrane, F, is a complex
function of frequency
Intermediate
frequency
region
High frequency
region
f ()
Low
frequency
region
Re[f ()]
Im[f ()]
AC Frequency (Hz)
Cell Dielectrophoresis
Positive and Negative Dielectrophoresis
Cell-pair DEP and Field stretch
Cell electrofusion
Recovery of cells
Applications of Electro-fusion
• Produce immortal hybrid cells (hybridomas)
secreting therapeutic proteins: For in vitro
production of therapeutic products or
diagnostics.
Examples: Monoclonal antibodies, growth
factors, Cytokines etc.
•Transgenic organisms/animals, multiploid animals
•Animal cloning
Creating hybridomas by pairwise
electrofusion
Electro-disinfection
Electro-disinfection of surgical instruments
Electro-disinfection of biologically contaminated
solutions by electroporation using only pulsed fields
is not practical on any large scale.
Electro-disinfection using electro-permeabilisation in
conjunction with low levels of cytotoxic agents
In-flow Hybrid disinfection
Hydrodynamic and electric focusing
Dielectric Septum
Hydrodynamic
focussing to guide
microcells toUsing
the centreline focused AC electric
fields
Apertures in septum
Focussed
Electric
Field
Electro-disinfection Flow Chamber
Dieletric septum for
micro-focusing AC field
Flow through
electrodes
In-flow disinfection with Hypochlorite.
Electro-disinfection of E. Coli using 1 kHz , 600V pulses in
conjunction with Hypochlorite.
Results for a 15 minute exposure to Hypochlorite
Control
: 50% kill rate.
Electro-disinfection
: 85% kill rate
50% kill rate with a 15 minute exposure to Hypochlorite
Control requires
: 0.003 ppm
Electro-disinfection
: 0.0003 ppm
Acknowledgements
Collaborators in research on electrical breakdown
and electroporation
Terry Chilcott (USYD, UNSW)
Ellie Gorczynska (UNSW)
Sue Murray-Jones (UNSW)
Agnus von Keller (Bonn)
Jane Taylor-Flemings (USYD)
David Monaghan (FuCell)
Lynn Oliver (RNSH)
Michael East (USYD)
Leonard Coster (UNSW)
Lutz Gaedt (UNSW)
Pikul Wanichapichart (PSU)
Tohsak Mahaworasilpa (UNSW)
John Smith (UNSW)
Heide Schnabl (Bonn)
John Kavanagh (USYD)