Micromachined Capillary Arrays for DNA Injection

用于 DNA 注射的微机械毛细管阵列

• 批准号: 11450100
• 负责人: FUJITA Hiroyuki
• 金额: $ 9.73万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11450100/
• 关键词: micromachine; microinjection; DNA injection; cell; biotechnology; genetic engineering

The purpose of this research is to fabricate micro capillary arrays for DNA injection to living cells by using micromachnining technology. The injection device is composed of the regular array of capillaries. Capillaries were pushed against cells and pierced them. Genetic substances such as DNA can be injected into cells through hollow capillaries. Capillaries are typically 1-5 micrometers in diameter, 0.5-1 micrometers in thickness, 20-50 micrometers in height and a few tens of micrometers in pitch between them. Major achievements of the research are :(1) We established the micromachining process for the capillary array. Thin holes of 5 micrometers in diameter and 100-200 micrometers in depth were etched in the silicon substrate by ICP-RIE (inductive coupled plasma-reactive ion etching). Then the inner surface of holes was thermally oxidized (0.5-1 micrometers in thickness). Etching of the silicon substrate from the backside revealed the bottom holes. The oxide layer at the bottom was removed by the short etching of buffered HF to make an open capillary. Further etching of the silicon substrate made capillaries protrude for 20-30 micrometers in height. Finally, a glass cover was anodically bonded on the top surface serving as a liquid reservoir and an external connector to a pipe and a pump.(2) The fabricated device was set against cultured tobacco cells. DNA solution was injected through piercing capillaries. We confirmed the production of a coded enzyme in cells.(3) We also fabricated a cell-capturing device to hold cells in a regular array with the same pitch of the injection device. The device had thinner through holes than the cell diameter. Cells suspended in liquid were captured on the holes by sucking the liquid through them from the backside.

本研究旨在利用微机械加工技术制备用于活体细胞DNA注射的微毛细管阵列。注射装置由规则排列的毛细管组成。将胶囊推到细胞上并刺穿它们。DNA等遗传物质可以通过中空的毛细血管注入细胞。电容器的直径通常为1-5微米，厚度为0.5-1微米，高度为20-50微米，它们之间的间距为几十微米。研究的主要成果有：（1）建立了毛细管阵列的微加工工艺。通过ICP-RIE（电感耦合等离子体反应离子蚀刻）在硅衬底中蚀刻直径为5微米且深度为100-200微米的薄孔。然后将孔的内表面热氧化（厚度为0.5-1微米）。从背侧蚀刻硅衬底揭示了底部孔。通过缓冲HF的短蚀刻去除底部的氧化物层以形成开放毛细管。进一步蚀刻硅衬底使毛细管突出20-30微米的高度。最后，将玻璃盖阳极结合在顶部表面上，用作液体储存器和与管道和泵的外部连接器。(2)制造的装置针对培养的烟草细胞设置。DNA溶液通过穿刺毛细管注射。我们证实了细胞中编码酶的产生。(3)我们还制作了一个细胞捕获装置，以保持细胞在一个规则的阵列与注射装置的相同的间距。该装置具有比单元直径更薄的通孔。悬浮在液体中的细胞通过从背面吸取液体而被捕获在孔上。

项目成果

Isao Kobayashi, Mitsutoshi Nakajima, Kyoseok Chun, Yuji Kikuchi and Hiroyuki Fujita: "Fabrication of Through-Type Microchannels for Monodiepersed Microspheres"Proceedings of NFRI-BRAIN International Workshop on Monodisperse Microspheres and Microchannel T

Isao Kobayashi、Mitsutoshi Nakajima、Kyoseok Chun、Yuji Kikuchi 和 Hiroyuki Fujita：“Fabrication of Through Type Microchannels for Monodiepersed Microspheres”NFRI-BRAIN 单分散微球和微通道 T 国际研讨会论文集

Hiroyuki Fujita: "Micromachined Tools for Nano and Bio Technologies"Korean MEMS Fair 2000 Seminar. 1-11 (2000)

Hiroyuki Fujita：“纳米和生物技术的微机械工具”2000 年韩国 MEMS Fair 研讨会。

B.Le Pioufle, P.Surbled, H.S.Chun, H.Fujita, H.Nagai, Y.Murakami, E.Tamiya: "Bio-Microsystem for Living Cells Manipulation : Application to the Gene Transfection"NAIR workshop, Tsukuba, Japan. (1999)

B.Le Pioufle、P.Surbled、H.S.Chun、H.Fujita、H.Nagai、Y.Murakami、E.Tamiya：“活细胞操作的生物微系统：在基因转染中的应用”NAIR 研讨会，日本筑波。

Kyoseok Chun,Gen Hashiguchi,Hiroshi Toshiyoshi,Hiroyuki Fujita, et al.: "An array of hollow microcapillaries for the controlled injection of genetic materials into animal/plant cells"IEEE Workshop on MEM S. 406-411 (1999)

Kyoseok Chun、Gen Hashiguchi、Hiroshi Toshiyoshi、Hiroyuki Fujita 等人：“用于将遗传物质受控注射到动物/植物细胞中的中空微毛细管阵列”IEEE Workshop on MEM S. 406-411 (1999)

Kyoseok Chun, Gen Hashiguchi, Hiroshi Toshiyoshi, Hiroyuki Fujita et al.: "Fabrication of Array of Hollow Microcapillaries Used for Injection of Genetic Materials into Animal/Plant Cells"Japan.J.Appl.Phys.. Vol.38. L279-281 (1999)

Kyoseok Chun、Gen Hashiguchi、Hiroshi Toshiyoshi、Hiroyuki Fujita 等人：“用于将遗传物质注射到动物/植物细胞中的中空微毛细管阵列的制作”Japan.J.Appl.Phys. 第 38 卷。