Dual Quantification of MicroRNAs and Telomerase in Living Cells
Dual Quantification of MicroRNAs and Telomerase in Living Cells
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活细胞中 MicroRNA 和端粒酶的双重定量
DOI：
10.1021/jacs.7b03617
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发表时间：
2017-08-30
影响因子：
15
通讯作者：
Xu, Chuanlai
中科院分区：
文献类型：
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作者：
Ma, Wei;Fu, Pan;Xu, Chuanlai
开发一种独特而通用的策略来同时定量活细胞中不同类型的生物分子(即核酸和蛋白质)是非常具有挑战性的。本文报道了一种基于表面增强拉曼散射和上转换的双信号平台，用于活细胞中microRNA(MiR)-21和端粒酶的超灵敏和原位定量检测。在miR-21和端粒酶存在下，miR-21与分子信标杂交导致分离。3，3‘-二乙基硫代氨基甲基菁碘修饰的Au-NR二聚体的拉曼信号减弱。此外，靶标端粒酶触发端粒酶引物链的延长，随后是置换杂交和上转换纳米颗粒的释放，导致发光增加。结果表明，细胞内miR-21的拉曼强度与其对数浓度在0.021~22.36amol/ng之间呈良好的线性关系，检出限为0.011 amol/ng。发光数据显示，细胞内端粒酶的对数浓度在0.6×10(-12)~31×10(-1)2IU之间呈线性关系，LOD为3.2×10(-13)IU。这些结果与拉曼成像和共聚焦成像符合得很好。重要的是，由于强烈的等离子体“热点”，miR-21的超灵敏检测成为可能。这种创新的双信号方法可以用于定量和精确地检测活细胞中的多种类型的信号分子，并了解细胞系统内的化学及其在疾病诊断中的应用。
The development of a unique and universal strategy for the simultaneous quantification of different types of biomolecules (i.e., nucleic acids and proteins) in living cells is extremely challenging. Herein, a two-signal platform, based upon surface enhanced Raman scattering and upconversion, for the ultrasensitive and quantitative in situ detection of microRNA (miR)-21 and telomerase in living cells is reported. In the presence of miR-21 and telomerase, the hybridization of miR-21 with a molecular beacon leads to the separation. of 3,3'-diethylthiocarbamyl cyanine iodide-modified Au NR dimers, resulting in a decrease in Raman signal. Also, the target telomerase triggers elongation of the telomerase primer strands, followed by substitutional hybridization and release of upconversion nanoparticles, leading to an increase in luminescence. A linear relationship between the Raman intensities and logarithmic concentration of intracellular miR-21 between 0.021 and 22.36 amol/ng(RNA) is observed; and the limit of detection (LOD) was determined to be 0.011 amol/ng(RNA). The luminescence data show a linear response between 0.6 x 10(-12) and 31 X 10(-1)2 IU for logarithmic concentration of intracellular telomerase with a LOD of 3.2 x 10(-13) IU. These results are in good agreement with Raman and confocal imaging. Importantly, the ultrasensitive detection of miR-21 was possible due to strong plasmonic "hot spots". This innovative two-signal approach can be utilized for the quantitative and precise detection of many types of signaling molecules in living cells and to understand the chemistry within cellular systems and its application in the diagnosis of disease.