Lifetime-based multiplexed detection of viral RNA using fluorogenic aptamers

probes targeting viral RNA fragments derived from SARS-CoV-2, MERS-CoV, and influenza A were designed and tested. Phasor plot analysis confirmed that these probes can robustly distinguish individual targets as well as mixtures containing any two or all three targets purely based on distinct fluorescence lifetimes of probes, thereby overcoming the challenges of cross-reactivity and spectral overlap. Beyond this proof of concept, our findings establish a generalizable strategy for engineering FAPs with customized photophysical properties, opening new avenues for next-generation diagnostics and molecular sensing technologies.