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.