Design and realization of a microfluidic device devoted to the application of ultra-short pulses of electrical field to living cells

In this paper, we present a novel microfluidic system dedicated to the application of ultra short pulses (i.e. nanopulses) on cells and the visualization of their effects. Cell plasma membranes can be rendered permeable by the use of nanosecond pulsed electric field. In conventional macroscopic electroporation chambers, the typical pulse duration is on the order of several hundred microseconds (micropulses), which allows the penetration of genes or other therapeutic molecules inside the cell. Consequently, electroporation pulses can be used in various clinical applications such as skin cancer treatment. A huge interest recently appeared on the use of ultra-short electric pulses (few nanoseconds) for the treatment of cells, as it was shown to enhance the treatment efficacy compared to micropulses. In this paper, the design, fabrication and biological validation of a microfluidic biochip optimized for the nanoporation of cells is detailed. The developed system presents several advantages such as real time monitoring of the effects of nanoporation on cells, single cell analysis, local generation of extremely high strength electrical fields while using reasonably low voltages and the precise control of electrical and fluidic parameters. To achieve the required spatial homogeneity of the electric field within the fluidic channel, the biochip includes thick electroplated gold electrodes (t=630 μm). As demonstrated in the paper, the electrical connections between the chip and the nsPEF generator are of prime importance, as it affects directly the quality of the impedance matching i.e. the transfert of energy between the generator and the cells. A study of the effects of these electrical connections on the biochip impedance is detailled. Nanoporation was achieved on living cells within the proposed chip, and characterized through the measurement of the fluorescence decay of intracellular calcein.