Miniaturized electroporation system for gene transfer using bio-MEMS technology.

摘要

Electroporation (EP) is currently a widely used bio-technique for delivery of drugs, or macromolecules to cells and tissues, which employs electrical pulse applied across a cell for cell membrane permeabilization. In this study, two-dimensional and three-dimensional array-type micro electroporation cell chips were fabricated using MEMS technology and tested with two mammalian cell lines. For the first time, a detailed parametric study of micro electroporation was conducted at the single-cell level. By quantitatively comparing the loading efficiency of large molecules and gene transfection on the 2D and 3D designs, we found that 3D electrodes design achieved a remarkable improvement.;Molecular size effect was then studied on 3D microchips by using five kinds of fluorescence labeled dextrans with different molecular weights. Extensive statistical data of the critical voltage and pulse duration were determined to construct an EP "phase diagram", which delineates the boundaries for (1) effective EP of five different size molecules and (2) electric cell lysis at the single-cell level. The resultant precise phase diagram can be very useful for the design of integrated micro systems and high throughput analysis. Comparing to the traditional instrument, micro electroporation chip can greatly shorten the experimental time.;The permeability of cell membrane was also measured by current response on micro EP chips. A nonlinear equivalent circuit model was proposed to describe the dynamic response of the whole system based on Electrochemical impedance spectroscopy (EIS). Using such a method, micro EP current directly related to the electropores was isolated from undesired leakage current to study the corresponding electropore dynamics. This novel method can circumvent the limitation of a regular fluorescence microscope.;Finally, a battery-powered signal generator with adjustable pulse amplitude, duration and pulse number was designed and fabricated by using an MSP430 microcontroller to realize a miniaturized, portable electroporation system.