IEEE Trans Nanobioscience. 2018 Mar;17(1):62-69. doi: 10.1109/TNB.2018.2798582.
Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.
电穿孔是一种将基因组分子导入选择性活细胞的物理转染方法，可以通过微电极设备来实现。较细的电极产生的局部电场可以在电极附近诱导细胞质膜穿孔。为了使用精细、高速扫描的电极，我们开发了一种精细的虚拟阴极图案，它是通过倒置电子束光刻技术在 100nm 厚的氮化硅（SiN）膜的细胞粘附表面上生成的。SiN 膜既可以作为真空屏障，也可以作为虚拟阴极的显示屏。入射的初级电子的动能完全被阻挡在 SiN 膜上，而在介电 SiN 膜的表面上，在 100nm 的区域内出现了负电荷和漏电电流。在培养介质中，我们在附着的 C2C12 成肌细胞上演示了局部控制的跨膜分子传递，其中含有荧光染料碘化丙啶（PI）。预稀释的 PI 的荧光增加表明在虚拟阴极位置发生了局部穿孔和跨膜流入，以及细胞内扩散。跨膜流入取决于束持续时间和加速电压。在分子传递后，细胞内 PI 荧光强度的轻微下降表明细胞膜从穿孔中恢复。通过暴露后细胞迁移的延时细胞成像来确认细胞活力。
