Nolkrantz K, Farre C, Brederlau A, Karlsson R I, Brennan C, Eriksson P S, Weber S G, Sandberg M, Orwar O
Department of Chemistry, Göteborg University, Sweden.
Anal Chem. 2001 Sep 15;73(18):4469-77. doi: 10.1021/ac010403x.
We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-microm o.d., 30-microm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.
我们展示了一种与高压电源相连的充满电解质的毛细管（EFC）如何用作一种通用的电穿孔工具，用于将染料、药物和生物分子递送至单细胞以及组织中细胞的细胞质。在EFC（熔融石英，长30 cm，外径375μm，内径30μm）上施加的大电压脉冲会在EFC末端外部产生一个小电场，这会导致细胞膜形成孔并诱导电解质的电渗流。当EFC含有细胞加载剂时，电渗流会在孔形成的部位输送这些试剂。孔形成和输送的结合使得物质能够加载到细胞质中。通过膜片钳和荧光显微镜观察到，在估计跨膜电压<85 mV时形成孔，半最大值约为206 mV。通过将荧光染料引入单个NG108 - 15细胞、细胞过程以及器官型海马培养物中的小细胞群体，证明了电穿孔方案。展示了初步结果，其中该方案用于大鼠脑腹侧中脑的体内电穿孔。该技术还用于进入细胞内基于细胞器的检测系统。作为一个示例，将1,4,5 - 肌醇三磷酸添加到电解质中，并通过电穿孔细胞中的细胞内细胞器进行检测。
