Aptamer-based flares hybridized with single-stranded DNA-conjugated MoS nanosheets for ratiometric fluorescence sensing and imaging of potassium ions and adenosine triphosphate in human fluids and living cells.

Addressing the limitations observed in previous studies, where the quantitative range of nanoprobes for detecting K and adenosine triphosphate (ATP) did not cover concentrations found within living cells, the present study aimed to develop ratiometric nanoprobes that can accurately sense changes in K and ATP levels in living cells and quantify them in human fluids. The proposed nanoprobes consisted of recognition flares modified with 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA), along with thiolate single-stranded DNA (ssDNA) and molybdenum disulfide nanosheets (MoS NSs). The thiolate ssDNA acts as a linker between the flares and the MoS NSs, directly forming a functional nanostructure at room temperature. The direct conjugation of labeled flares to the MoS NSs simplifies the fabrication process. In the absence of K and ATP, the hybridization of flares and thiolate ssDNA caused FAM to move away from TAMRA, suppressing the fluorescence resonance energy transfer (FRET) process. However, upon the introduction of K and ATP, the flares undergo a structural transformation via the formation of G-quadruplex formation and the generation of hairpin-shaped structures, respectively. This structural change leads to the release of the flares from the ssDNA-conjugated nanosheet surface. The release of the flares brings FAM and TAMRA into close proximity, allowing FRET to occur, leading to FRET and static quenching. By monitoring the ratio between the fluorescence intensities of FAM and TAMRA, the concentration of K (5-100 mM) and ATP (0.3-5 mM) can be accurately determined by the proposed nanoprobes. The advantages of these nanoprobes lie in their ability to provide ratiometric measurements, which enhance the accuracy and reliability of the quantification process. The proposed nanoprobes offer potential applications as ratiometric imaging probes for monitoring K and ATP-related reactions in living cells, providing valuable insights into cellular processes. Additionally, they can be employed for determining the levels of K and ATP in human fluids, offering potential diagnostic applications in various clinical settings.