Rapid, facile, reagentless, and room-temperature conjugation of monolayer MoS2 nanosheets with dual-fluorophore-labeled flares as nanoprobes for ratiometric sensing of TK1 mRNA in living cells

Abstract

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 (DNA_pm) 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 DNA_pm 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.