(102c) Electrorotation As a Tool to Study Interaction Kinetics Between Proteins and Cells in Real-Time

Electrorotation
as a tool to study interaction kinetics between proteins and cells in real-time

Samuel
Kilchenmann*,
Fabio Spiga* and Carlotta Guiducci*

*École Polytechnique
Fédérale de Lausanne, SWITZERLAND

Introduction

Electrorotation
has been used to assess electrical parameters of biological particles such as
cells, therefore allowing to distinguish between e.g. viable and
non-viable yeast cells [1]. In biological sciences the evolution of cellular
parameters over time is also very informative, for instance to determine the kinetics
of protein-cell interactions in real-time. Furthermore, the possibility to simultaneously
observe phenotypic changes of the cells allows to correlate them with the
electrokinetic data. In the present work, we propose an electrorotation setup coupled
with high resolution fluorescence imaging to observe the particle's movements.
The bulky size and the short working distance of the conventional microscope lenses,
which are normally used for high resolution fluorescence imaging, are critical
for the fabrication of the electrorotation chips. For this reason, we developed
a fabrication process that allows aligning microfluidic channels made in SU-8 with
photolithographic accuracy with a glass top coverage, and featuring backside
microfluidic accesses. Furthermore, these chips include 3D metal electrodes
providing a high linearity of the rotating electric field, without any field
component in the z-axis. These electrodes are advantageous to focus and follow
single particles over time, since the particles are not moved out of the focal
plane by dielectric forces resulting from the z-components of the electric
field, as it commonly happens with planar electrodes.

Microfabrication

The
fabrication of the electrorotation chips starts by defining the microfluidic
accesses with standard photolithography and transferring the pattern
300 μm deep into a passivated silicon substrate. Then, the 3D
structures are photopatterned in SU-8 that is afterwards covered with a metal
layer by sputter deposition. Then, a dry film resist is applied and structured and
it serves as etch mask for the metal layer. A further step of SU-8 lithography
is done to define the microfluidic channels. In between the post exposure bake
and the development of the SU-8, the resist topography is smoothened out by
chemical mechanical polishing. After development of the second SU-8 layer, the
wafers are grinded from the backside down to 300 μm in order to open
up the microfluidic accesses. Finally, the wafers are diced and a microscopic
cover slip is thermally bonded on top of the microfluidic channel.

Fluorescence
imaging and electrorotation of healthy and apoptotic HEK cells

The
suitability of our system for high resolution imaging was tested by imaging non-fixed
healthy and apoptotic HEK cells. Apoptosis was induced through heat shock (45
minutes at 44°C) followed by a 2 hours recovery at 37°C. The cells were then
washed and stained in a solution containing sucrose (8.5%),  dextrose (0.3%) and
Acridine Orange (50 μg/ml). The conductivity of this solution was
adjusted to 0.1 mS by the addition of PBS. By imaging the cells, it was possible
to distinguish between healthy cells, showing a roundish and clear nucleus
(Figure 1a), and apoptotic cells, with deformed and non homogenously
stained nucleus (Figure 1b). Electrorotation experiments were then
performed by applying a signal with amplitude of 4-5 V_p-p at a
frequency of 100 kHz. In these experiments it was possible to correlate
the fluorescence imaging with the electrorotation data and to observe that healthy
cells have a different sense of rotation with respect to apoptotic cells. In
particular, apoptotic cells rotate in the direction of the electric field,
while healthy cells rotate in the opposite direction. The observed rotation of
the particles is fairly stable, making it possible to use this setup to study
the evolution of the particle's parameters in real-time over a extended time.

Figure
1: Fluorescence picture of normal (a) and apoptotic (b) HEK cells taken inside
a microfluidic channel

1.         Ying,
H., et al., Differences in the Ac Electrodynamics of Viable and Nonviable
Yeast-Cells Determined through Combined Dielectrophoresis and Electrorotation
Studies. Physics in Medicine and Biology, 1992. 37(7): p. 1499-1517.