Kerstin Nolkrantz

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.