Evaluation of the actin cytoskeleton state using an antibody-functionalized nanoneedle and an AFM
Evaluation of the actin cytoskeleton state using an antibody-functionalized nanoneedle and an AFM
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DOI：
10.1016/j.bios.2012.06.044
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发表时间：
2013-02-15
影响因子：
12.6
通讯作者：
Nakamura, Chikashi
中科院分区：
文献类型：
--
作者：
Silberberg, Yaron R.;Mieda, Shingo;Nakamura, Chikashi
开发了一种细胞诊断技术，其使用原子力显微镜（AFM）和削尖至200 nm直径的超薄AFM探针（纳米针）。由于纳米针的高纵横比，它被成功地插入到活细胞中，而不影响其活力。此外，通过用特异性抗体官能化纳米针并在纳米针从细胞中排空期间测量解结合力（“钓鱼力”），可以测量抗体官能化的纳米针与细胞的细胞内内容物之间的特异性机械相互作用。在这项研究中，抗肌动蛋白抗体功能化的纳米针被用来评估活细胞中的肌动蛋白细胞骨架状态。为了检查细胞骨架条件对测量的捕鱼力的影响，使用了细胞骨架破坏药物细胞松弛素D（cytD）和Y-27632，结果显示，与任何一种药物孵育后，测量的捕鱼力显着下降。此外，该技术被用来测量在与cytD孵育过程中的单细胞的时间过程的变化，显示了一个逐渐的时间依赖性下降的捕捞力量。即使是微小剂量的药物，其效果几乎不明显的光学和荧光方法，可以清楚地检测到纳米针蛋白质捕鱼力的测量，指出这种检测方法的高灵敏度。该技术可证明有益于评估健康和疾病中的细胞骨架状况，以及根据其细胞内蛋白质含量选择特定细胞，而不需要将标记蛋白引入细胞中。(c)2012 Elsevier B. V.保留所有权利。
A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell. (c) 2012 Elsevier B.V. All rights reserved.