Photoactivated Specific mRNA Detection in Single Living Cells by Coupling "Signal-on" Fluorescence and "Signal-off" Electrochemical Signals
Photoactivated Specific mRNA Detection in Single Living Cells by Coupling "Signal-on" Fluorescence and "Signal-off" Electrochemical Signals
复制标题
DOI：
10.1021/acs.nanolett.8b02004
复制
发表时间：
2018-08-01
期刊：
影响因子：
10.8
通讯作者：
Willner, Itamar
中科院分区：
文献类型：
--
作者：
Huang, Fujian;Lin, Meihua;Willner, Itamar
单个活细胞中靶mRNA的时空检测是纳米科学和纳米医学的主要挑战。我们介绍了一种在单个活细胞水平上检测mRNA的通用方法，该方法使用光切割发夹探针作为功能单元，用于单个MCF-7癌细胞中MnSOD mRNA的光学（荧光）和电化学（伏安）检测。荧光探针由邻硝基苯基磷酸酯功能化发夹组成，包括FAM荧光团，其呈笼状结构，经Dabcyl猝灭。利用AS 1411适体对荧光探针进行进一步修饰，以促进探针靶向和内化到MCF-7细胞中。在紫外线照射下，发夹被切割，导致细胞内fam功能化链的脚点刺激位移，从而在单个细胞中检测到mRNA时产生开关荧光信号。此外，用亚甲基蓝（MB）氧化还原活性光切割发夹功能化的纳米电极被插入单个MCF-7细胞的细胞质中。发夹的光裂解导致与探针相关的氧化还原活性链的mrna介导的支点位移，导致探针伏安响应的耗尽。平行光学和电化学检测mRNA在单细胞水平被证明。
The spatiotemporal detection of a target mRNA in a single living cell is a major challenge in nanoscience and nanomedicine. We introduce a versatile method to detect mRNA at a single living cell level that uses photocleavable hairpin probes as functional units for the optical (fluorescent) and electrochemical (voltammetric) detection of MnSOD mRNA in single MCF-7 cancer cells. The fluorescent probe is composed of an ortho-nitrophenylphosphate ester functionalized hairpin that includes the FAM fluorophore in a caged configuration quenched by Dabcyl. The fluorescent probe is further modified with the AS 1411 aptamer to facilitate the targeting and internalization of the probe into the MCF-7 cells. Under UV irradiation, the hairpin is cleaved, leading to the intracellular mRNA toehold-stimulated displacement of the FAM-functionalized strand resulting in a switched-on fluorescence signal upon the detection of the mRNA in a single cell. In addition, a nanoelectrode functionalized with a methylene blue (MB) redox-active photocleavable hairpin is inserted into the cytoplasm of a single MCF-7 cell. Photocleavage of the hairpin leads to the mRNA-mediated toehold displacement of the redox-active strand associated with the probe, leading to the depletion of the voltammetric response of the probe. The parallel optical and electrochemical detection of the mRNA at a single cell level is demonstrated.