ABSTRACT 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.
Xu et al. (2026) studied this question.