Claire Dalmay

A microfluidic biochip for the nanoporation of living cells

This paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3-10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3 ns and 10 ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells.

A microfluidic device with removable packaging for the real time visualisation of intracellular effects of nanosecond electrical pulses on adherent cells

The biological mechanisms induced by the application of nanosecond pulsed electric fields (nsPEFs: high electrical field amplitude during very short duration) on cells remain partly misunderstood. In this context, there is an increasing need for tools that allow the delivering of such pulses with the possibility to monitor their effects in real-time. Thanks to miniaturization and technology capabilities, microtechnologies offer great potential to address this issue. We report here the design and fabrication of a microfluidic device optimized for the delivery of ultra short (10 ns) and intense (up to 280 kV cm(-1)) electrical pulses on adherent cells, and the real time monitoring of their intracellular effects. Ultra short electric field pulses (nsPEFs or nanopulses) affect both the cell membrane and the intracellular organelles of the cells. In particular, intracellular release of calcium from the endoplasmic reticulum was detected in real time using the device, after exposure of adherent cells to these nsPEFs. The high intensity and spatial homogeneity of the electric field could be achieved in the device thanks to the miniaturization and the use of thick (25 μm) electroplated electrodes, disposed on a quartz substrate whose transparency allowed real time monitoring of the nsPEFs effects. The proposed biochip is compatible with cell culture glass slides that can be placed on the chip after separate culture of several days prior to exposure. This device allows the easy exposure of almost any kind of attached cells and the monitoring in real time while exposed to nsPEFs, opening large possibilities for potential use of the developed biochips.