Han Sung-Woong, Ryu Seunghwan, Kitagawa Taro, Uetsuka Hiroshi, Fujimori Naoji, Aoki Yukihiro, Ota Ryo, Amemiya Yosuke, Shimamoto Nobuo, Nakamura Chikashi, Miyake Jun
Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), Central 6 Tsukuba, Ibaraki, Japan.
Arch Histol Cytol. 2009;72(4-5):261-70. doi: 10.1679/aohc.72.261.
We have been developing a low invasive cell manipulation technology based on inserting an ultra-thin needle--"nanoneedle"--into a living cell by using an atomic force microscope (AFM). The nanoneedle, made from a silicon AFM tip by focused-ion-beam etching, has a diameter of several hundred nanometers and a length of about 10 microns. Successful insertion of the nanoneedle into the cell can be confirmed by the appearance of a steep relaxation of repulsive force in the force-distance curve as monitored by the AFM system. This technology, termed "cell surgery", can be applied for the detection of intracellular proteins in a living cell or for highly efficient gene transfer. The present study shows that the durability of a tapered nanoneedle is superior to that of a cylindrical nanoneedle, and that a proper aspect ratio for the tapered nanoneedle must be chosen to maintain sufficient insertion efficiency for a particular target cell: tapered nanoneedles of an aspect ratio over 20 showed high insertion efficiency for various kinds of mammalian cells. We then used diamond for the material of the nanoneedle because its specific properties, such as high stiffness, heat conductivity, and electrical conductivity capacitated by boron doping, were deemed useful for the analysis and manipulation of intracellular phenomena. We compared the capability of the diamond nanoneedle in cell manipulation with that of the silicon nanoneedle. Evaluation of the effect of the former on transcription efficiency and localization analysis of p53 expression revealed the low invasiveness for cell manipulation as was also the case for the silicon nanoneedle. We also succeeded in achieving highly efficient plasmid DNA delivery into a mouse fibroblast C3H10T1/2 using the diamond nanoneedle. The diamond nanoneedle is expected to contribute to the versatility of "cell surgery" technology.
我们一直在开发一种基于使用原子力显微镜（AFM）将超薄针——“纳米针”——插入活细胞的低侵入性细胞操作技术。这种纳米针由硅AFM针尖通过聚焦离子束蚀刻制成，直径为几百纳米，长度约为10微米。通过AFM系统监测力-距离曲线中排斥力的急剧松弛现象，可以确认纳米针成功插入细胞。这项技术被称为“细胞手术”，可用于检测活细胞内的蛋白质或进行高效的基因转移。本研究表明，锥形纳米针的耐用性优于圆柱形纳米针，并且必须为锥形纳米针选择合适的纵横比，以保持对特定靶细胞的足够插入效率：纵横比超过20的锥形纳米针对各种哺乳动物细胞显示出高插入效率。然后，我们使用金刚石作为纳米针的材料，因为其特定性能，如高硬度、热导率以及通过硼掺杂获得的导电性，被认为对细胞内现象的分析和操作有用。我们将金刚石纳米针在细胞操作中的能力与硅纳米针进行了比较。对前者对转录效率的影响以及p53表达的定位分析表明，其在细胞操作中的侵入性较低，硅纳米针也是如此。我们还成功地使用金刚石纳米针将质粒DNA高效递送至小鼠成纤维细胞C3H10T1/2中。金刚石纳米针有望为“细胞手术”技术的多功能性做出贡献。
