Abstract
It has been observed that when certain electrical potentials are applied across a cell they can induce the formation of pores in the cell membrane and consequently increase the permeability of the cell to macromolecules. This phenomenon is known as electroporation. Since the first report on gene transfer by electroporation1, it has become a standard method for introduction of macromolecules into cells2 3 4. Currently, electroporation is normally done in batches of cells between electrodes and there is little control over the permeabilization of individual cells. Therefore, it is very difficult to study the fundamental biophysics of cell membrane electro-permeabilization, which is not yet understood, and to design optimal and reversible electroporation protocols for individual cells2 3. Although the biophysics of electroporation are still not fully understood, indirect evidence shows that micro aqueous pores with diameters of tens to hundreds of angstroms are created in cell membrane due to the electrical field induced structural rearrangement of the lipid bilayer5.
It occurred to us that if electroporation induces pores in the cell membrane than, in a state of electroporation, a measurable current should flow through the individual cell. From this idea, we have developed a new micro-electroporation technology that employs a “bionic” chip to study and control the electroporation process in individual cells. The micro-electroporation chips are designed and fabricated using standard silicon microfabrication technology. Figure 1 shows the schematic of the chip in cross section. Each chip is a three-layer device that consists of two translucent poly silicon electrodes and a silicon nitride membrane, which all together form two fluid chambers. The two chambers are interconnected only through a micro hole on the dielectric silicon nitride membrane. In a typical process, the two chambers are filled with conductive solutions and one chamber contains biological cells. Individual cells can be captured in the micro hole and thus incorporated in the electrical circuit between the two electrodes of the chip. When the cell is in its normal state no current flows through the insulating lipid bilayer and consequently between the electrodes. However, when the electrical potential across the electrodes is sufficient to induce electroporation, a measurable current will flow through the pores of the cell membrane and between the electrodes. Measuring currents through the bionic chip as a function of electrical potential will determine the potential that induces the electroporation. The chip behaves somewhat similarly to an electrical diode, with no current at potentials that do not induce electroporation and currents at potentials that induce electroporation. With the ability to manipulate individual cells and detect the electrical potentials that induce electroporation in each cell, the chip can be used to study the fundamental biophysics of membrane electro-permeabilization on single cell level and in biotechnology, for controlled introduction of macromolecules, such as gene constructs, into individual cells. We anticipate that this new technology will change the way in which electroporation is done and will provide key understanding of the biophysical processes that lead to cell electroporation.
In this paper, first the design, fabrication process and modeling of the microelectroporation chip are described in details. Subsequently, experiment methods and results are presented and discussed, demonstrating the feasibility of altering cell membrane permeability and facilitating intercellular mass transfer in a more controlled way on single cell level. Finally, the potential applications of the micro-electroporation chips and future research directions are discussed.
Figure 2 demonstrates how cell membrane electroporation can be investigated through monitoring and analyzing chip current-voltage signatures.
Signaling mechanism by the Staphylococcus aureus two-component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS