Biomedical Engineering Reference
In-Depth Information
homogeneous electric fields across fixed distances of 0.1-
5 mm, several times larger than the size of a single
mammalian cell (Ryttsen et al., 2000). Also there are
numerous experimental methods for the biochemical and
biophysical investigations of single cells. Such methods
include (1) patch clamp techniques for measuring trans-
membrane currents through a single ion channel (Hamill
et al., 1981), (2) scanning confocal and multi-photon
microscopy for imaging and localizing bioactive compo-
nents in single cells and single organelles (Maiti et al.,
1997), (3) near-field optical probes for measuring pH in
the cell interior (Song et al., 1997), (4) ultra-
microelectrodes for monitoring the release of single cat-
echol- and indol-amine-containing vesicles (Wightman
et al., 1991; Chow et al., 1992), (5) optical trapping and
capillary electrophoresis separations for analyzing the
chemical composition of individual secretory vesicles
(Chiu et al., 1998), (6) electroporation with solid micro-
electrodes (Lundqvist et al., 1998), (7) electroporation
with capillaries and micropipettes (Haas et al., 2001;
Nolkrantz et al., 2001, Rae and Levis, 2002), (8) micro-
fabricated chips and multiplexed electroporation system
(Huang and Rubinsky, 1999; Lin , 2001).
Rubinsky's group presented the first microfluidic
device to electroporate a cell (Huang and Rubinsky,
1999; Davalos et al., 2000). Their device consisted of
three silicon chips bonded together to form two cham-
bers, separated by a 1 m m thick silicon nitride membrane
with a 2-10 m m diameter hole. Since silicon nitride is
non-conductive, any electrical current flowing from the
top chamber to the bottom chamber must pass through
this microhole. A cell suspension was introduced into the
top chamber, followed by the immobilization of one cell
in the hole by lowering the pressure in the bottom
chamber. Since the trapped cell impedes the system's
electrical path, only low voltage pulses are needed to
induce large fields near the trapped cell and only the
trapped cell is electroporated. With this chip, they were
able to show the natural difference in electroporation
behavior between human prostate adenocarcinoma and
rat hepatocyte cells by studying the process using
current-voltage measurements (Huang and Rubinsky,
1999). Davalos and colleagues advanced this technology
by making the chambers and electrodes off-chip, sim-
plifying it to one silicon chip. Such changes enabled ease
of use, accessibility of the device and reusability (Lee
et al., 2006, Robinson et al., 2007).
In recent years, several microfluidic electroporation
designs for the analysis, transfection or pasteurization of
biological cells have been reported (Fox et al., 2006a,b).
The range of applications for microfluidic electroporation
coupled with advances in microfabrication techniques,
specifically the use of structural photoresist for soft li-
thography, has resulted in a variety of designs: microchips
in which cells move through a treatment zone (Gao et al.,
2004), microchips in which cells are trapped at a specific
location (Huang and Rubinsky, 1999) and devices in
which the cells are surface-bound (Lin and Huang, 2001;
Fox et al., 2006a,b). Of all the types of designs created,
only the few designs in which a cell is trapped at a specific
location enables us to study the biophysics of electro-
poration at the single cell level. In addition to the original
devices described in the previous paragraph, other designs
have been developed in which a cell is trapped at a specific
location and electroporated. For example, Huang and
Rubinsky advanced their technology using structural
photoresist to create microfluidic channels on top of their
silicon wafer (Huang and Rubinsky, 2003). Khine et al.
fabricated a device using soft lithography which was
originally developed as amultiple patch clamp array. Their
device contains a main channel with multiple perpen-
dicular small side channels. The individual cells in the
main channel are brought into contact with the opening of
a side channel using pressure. The cell does pass the
constriction because its diameter (12-17 m m) is approx-
imately 4 times larger than the constriction (3.1 m m). The
constriction enables potentials of less than 1V to deliver
the high fields needed to induce electroporation, which is
applied using an silver-silver chloride-electrode (Khine
et al., 2005a,b). Such devices are useful to study the
biophysical process of electroporation because the
changes in electrical properties of an individual cell as well
as the molecular transport into the cell can be tracked
(Davalos et al., 2000).
Supraelectroporation
If the applied electric field is very high ( > 10 kV/cm) and
the pulses are very short (nanaosecond range), not only
the plasma membrane of a cell is rendered permeable but
also intracellular structures (Schoenbach et al., 2004;
Vasilkoski et al., 2006). This opens new perspectives for
treatment of cells, especially involving intracellular
structures.
Electroporation theoryworks well up to about 2 kV/cm
applied electric field. At higher field strength some
effects appear which are hardly explainable just by pore
formation.
The cell membrane has a capacity on the order of
1 m F/cm 2 . At 30 MHz its capacitive resistance is small
compared to the resistance of the electrolytes (i.e. the
membranes are de facto shortened). The conductivity at
this frequency is therefore a good guide for the maxi-
mum extent of electroporation, which would be when all
membranes contain 100% pores. Even if it is practically
irrelevant, it gives an idea about the absolute maximum
of conductance. As shown in Fig. 4.1-21 , the conduc-
tance exceeds this maximum value considerably when
the field exceeds about 5 kV/cm. An early explanation
involved some kind of Wien effect. The first Wien effect
Search WWH ::




Custom Search