Self‐Calibrating Ratiometric DNA Aptamer Probe for Quantitative ATP Imaging in Living Cells

Accurate monitoring of adenosine triphosphate (ATP)-the universal energy currency of cells-is essential for elucidating cellular metabolism and disease progression. However, the high basal concentration of intracellular ATP (1-10 mM) and variable probe uptake during imaging have hampered the development of reliable fluorescent sensors. Although DNA aptamer-based probes provide excellent selectivity, conventional turn-on designs often lack internal calibration, and Förster resonance energy transfer-based ratiometric probes typically exhibit limited signal changes. We report a ratiometric DNA duplex sensor comprising a Cy5-labeled ATP aptamer and a thiazole orange (TO)-labeled exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probe. Upon ATP binding, the aptamer structure switches and releases the reporter strand, resulting in a pronounced decrease in TO fluorescence while the Cy5 signal remains constant. Rational insertion of a polythymidine spacer effectively suppressed undesired TO-to-Cy5 energy transfer, enabling a reliable ratiometric Cy5/ECHO readout. The sensor operates robustly across physiological ATP concentrations, exhibits high nucleotide selectivity and satisfactory serum stability, and shows minimal cytotoxicity. Live-cell flow cytometry and confocal imaging further confirmed that cancer cells displayed significantly higher Cy5/ECHO ratios than normal fibroblasts. This internally self-calibrating aptamer sensor thus provides a powerful platform for intracellular ATP imaging and cancer diagnostics.