College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Molecular and Nano Science, Shandong Normal University , Jinan 250014, P. R. China.
ACS Appl Mater Interfaces. 2018 Feb 28;10(8):6982-6990. doi: 10.1021/acsami.7b19284. Epub 2018 Feb 15.
In situ imaging of microRNA (miRNA) in living cells and in vivo is beneficial for promoting the studies on miRNA-related physiological and pathological processes. However, the current strategies usually have a low signal-to-background ratio, which greatly affects the sensitivity and imaging performance. To solve this problem, we developed a highly sensitive strategy for fluorescence imaging of miRNA in living cells and in vivo based on graphene oxide (GO)-enhanced signal molecule quenching of a molecular beacon (MB). 2Cy5-MB was designed by coupling two Cy5 molecules onto the opposite ends of MB. The fluorescence intensities of two Cy5 molecules were reduced because of the self-quenching effect. After adsorbing on the GO surface, the fluorescence quenching of the molecules was enhanced by fluorescence resonance energy transfer. This double-quenching effect significantly reduced the fluorescence background. In the presence of one miRNA molecule, the fluorescence signals of two Cy5 molecules were simultaneously recovered. Therefore, a significantly enhanced signal-to-background ratio was obtained, which greatly improved the detection sensitivity. In the presence of miRNA, the fluorescence intensity of 2Cy5-MB-GO recovered about 156 times and the detection limit was 30 pM. Compared with 1Cy5-MB-GO, the elevated fluorescence intensity was enhanced 8 times and the detection limit was reduced by an order of magnitude. Furthermore, fluorescence imaging experiments demonstrated that 2Cy5-MB-GO could visually detect microRNA-21 in various cancer cells and tumor tissues. This simple and effective strategy provides a new sensing platform for highly sensitive detection and simultaneous imaging analysis of multiple low-level biomarkers in living cells and in vivo.
在活细胞和体内原位成像 miRNA（miRNA）有益于促进 miRNA 相关生理和病理过程的研究。然而，目前的策略通常具有较低的信号背景比，这极大地影响了灵敏度和成像性能。为了解决这个问题，我们开发了一种基于氧化石墨烯（GO）增强分子信标（MB）信号分子猝灭的高灵敏度用于活细胞和体内 miRNA 荧光成像的策略。通过将两个 Cy5 分子连接到 MB 的相对末端，设计了 2Cy5-MB。两个 Cy5 分子的荧光强度由于自猝灭效应而降低。吸附在 GO 表面后，分子的荧光猝灭通过荧光共振能量转移得到增强。这种双重猝灭效应显著降低了荧光背景。在存在一个 miRNA 分子的情况下，两个 Cy5 分子的荧光信号同时得到恢复。因此，获得了显著增强的信号背景比，大大提高了检测灵敏度。在存在 miRNA 的情况下，2Cy5-MB-GO 的荧光强度恢复了约 156 倍，检测限为 30 pM。与 1Cy5-MB-GO 相比，荧光强度提高了 8 倍，检测限降低了一个数量级。此外，荧光成像实验表明，2Cy5-MB-GO 可以在各种癌细胞和肿瘤组织中可视化检测 microRNA-21。这种简单有效的策略为活细胞和体内多种低水平生物标志物的高灵敏度检测和同时成像分析提供了一个新的传感平台。
