Photoactivated Specific mRNA Detection in Single Living Cells by Coupling "Signal-on" Fluorescence and "Signal-off" Electrochemical Signals.

Abstract

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 AS1411 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.