Rapid, facile, reagentless, and room-temperature conjugation of monolayer MoS nanosheets with dual-fluorophore-labeled flares as nanoprobes for ratiometric sensing of TK1 mRNA in living cells.

Direct loading of fluorophore-labeled DNA molecules (named as flares) on gold nanoparticles (AuNPs) is a controllable and straightforward approach for intracellular imaging of target DNA molecules. However, the modification of AuNPs with flares requires a tedious and time-consuming procedure, additional reagents, or adenosine-rich DNA molecules. Here, we developed a rapid, simple, reagentless, and room-temperature approach for the modification of monolayer molybdenum disulfide nanosheets (M-MoS2 NSs) with dual-fluorophore-labeled flares, which were implemented for the ratiometric imaging of TK1 mRNA in living cells. The duplexes were prepared by hybridizing thiolated single-stranded DNA (ssDNA) to 6-carboxyfluorescein (FAM)- and 5-carboxytetramethylrhodamine (TAMRA)-labeled flares. The nanoflares were fabricated by conjugating the formed duplexes to the surface sulfur vacancy sites of the M-MoS2 NSs. The time required for preparing the nanoflares was found to be within 1 h. In the nanoflares, FAM stays away from TAMRA, leading to inefficient fluorescence resonance energy transfer (FRET). The presence of perfectly matched DNA (DNApm) molecules induces the liberation of the flares from the nanoflares. The liberated flares fold into hairpin-shaped structures, causing high FRET efficiency from FAM to TAMRA and efficient FAM-TAMRA static quenching. Following this mechanism, the nanoflares provided an effective platform for the ratiometric sensing of DNApm molecules with a limit of detection (at a signal-to-noise ratio of 3) of 8 nM and the linear range of 25-500 nM. Confocal microscopy experiments demonstrated that the nanoflares can be used to ratiometrically image TK1 mRA in HeLa and MCF-7 cells.