Development of a novel method to detect intrinsic mRNA in a living cell by using a molecular beacon-immobilized nanoneedle
Development of a novel method to detect intrinsic mRNA in a living cell by using a molecular beacon-immobilized nanoneedle
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DOI：
10.1016/j.bios.2010.07.079
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发表时间：
2010-12-15
影响因子：
12.6
通讯作者：
Miyake, Jun
中科院分区：
文献类型：
--
作者：
Kihara, Takanori;Yoshida, Narutoshi;Miyake, Jun
我们开发了一种新型的纳米生物传感器，通过配备纳米探针的原子力显微镜(AFM)来监测单个活细胞中的mRNA表达。通过中性亲和素将生物素修饰的分子信标固定在超细针(纳米针)上，构建了纳米探针。为了评价该纳米探针的有效性，我们选择了3-磷酸甘油醛脱氢酶(GAPDH)基因作为靶标。单个HeLa细胞含有约1000个拷贝的GAPDHmRNA。该纳米探针被直接插入活的HeLa细胞中，并与细胞内的固有靶向mRNA发生反应。通过将纳米探针从细胞中拔出，可以对其进行复性。此外，我们成功地利用该纳米探针在多个细胞中连续检测GAPDHmRNA，即该纳米探针对单个活细胞中固有的mRNA的检测具有高度的特异性和敏感性。MRNAs被认为是高度浓缩的，因为细胞中存在大量的细胞器和复合体，而可用于分布的空间有限。因此，直接分析活细胞中的内在RNA将是有利的，我们的新型纳米探针非常适合于监测活细胞中的RNA。(C)2010爱思唯尔B.V.保留所有权利。
We developed a novel nanobiosensor for monitoring mRNA expression in a single living cell by using an atomic force microscope (AFM) equipped with a nanoprobe. The nanoprobe was constructed by immobilizing a biotin-modified molecular beacon onto an ultrathin needle (nanoneedle) via neutravidin. In order to evaluate the effectiveness of the nanoprobe, we selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the target. A single HeLa cell contained approximately 1000 copies of GAPDH mRNA. The nanoprobe was directly inserted into living HeLa cells, and it reacted with the intrinsic target mRNA within the cells. The nanoprobe could be renatured by pulling it out of the cells. Further, we successfully used the nanoprobe for continuous detection of GAPDH mRNA in multiple cells, i.e., the nanoprobe was highly specific and sensitive for the detection of intrinsic mRNA in single living cells. mRNAs are thought to be highly condensed because of the large number of organelles and complexes present in cells and the limited space available for distribution. Therefore, direct analysis of intrinsic mRNAs in living cells would be advantageous, and our novel nanoprobe is highly suitable for monitoring the RNAs in living cells. (C) 2010 Elsevier B.V. All rights reserved.