Novel Single Bio-Molecule Measurement using Micro/Nano Machining Technology

使用微/纳米加工技术的新型单生物分子测量

• 批准号: 14205035
• 负责人: KIM Beomjoon
• 金额: $ 34.61万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14205035/
• 关键词: Bio-molecular motor; Micro; Nano machine; Nano machining technology; MEMS; Biophysics; Single molecule measurement; Nano patterning; マイクロ・ナノマシン

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. We provide here direct evidence that F1 is designed to tightly … More couple its catalytic reactions with the mechanical rotation. Our results suggest that the 1-subunit has an essential function during ATP synthesis. Moreover, we propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F1-ATPase biomolecular motors ; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the shadow mask. Less

F1-ATPase是每回合最小的旋转运动催化事件，与配合物的分子结构一致。 F1（顺时针）F1的旋转，导致ATP合成。与机械旋转的合成离子的速度与合成的量成比例。玻璃底物使用带有锥形孔的穿孔聚二甲基硅氧烷E通过在筛子上自旋涂层PDM，荧光异硫氰酸盐，牛） - 烯烃 - 烯烃 - 烯烃在阵列中发现了非特异性结合的问题。为了表明蛋白质维持其活性，我们使用了生物分子的活动；

项目成果

G.M.Kim, B.J.Kim, M.Liebau, J.Huskens, D.N.Reinhoudt, J.Brugger: "Surface modification with self-assembled monolayers for nanoscale replication of photoplastic MEMS"Journal of MEMS. Vol.11No.3. 175-181 (2002)

G.M.Kim、B.J.Kim、M.Liebau、J.Huskens、D.N.Reinhoudt、J.Brugger：“用于光塑 MEMS 纳米级复制的自组装单层表面改性”MEMS 杂志。

Hiroyuki Noji: "Single-molecule manipulation of F1-motor with magnetic tweezers"The 6th MEMBRANE RESERCH FORUM. 6

Hiroyuki Noji：“用磁力镊子对 F1 电机进行单分子操纵”第六届膜研究论坛。

chapter 5, Large area micro/nano patterning through Top-down and bottom-up approaches (a chapter contributed to book "Ultra high precision patterning technology-Next generation micro/nano process")

第五章，通过自上而下和自下而上的方法进行大面积微纳图案化（《超高精度图案化技术-下一代微纳工艺》一书的章节）

• 发表时间: 2006
• 作者: S.-K.Kim;et al.;Kim BJ.
• 通讯作者: Kim BJ.

Microfabricated arrays of femtoliter chambers allow single molecule enzymology

• DOI: 10.1038/nbt1072
• 发表时间: 2005-03-01
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Rondelez, Y;Tresset, G;Noji, H
• 通讯作者: Noji, H

Activation of pausing F1 motor by external force

• DOI: 10.1073/pnas.0406486102
• 发表时间: 2005-03-22
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hirono-Hara, Y;Ishuzuka, K;Noji, H
• 通讯作者: Noji, H