An entropy-driven DNA timer fluorescence sensor for one-pot sequential detection of three respiratory viruses.

Nucleic acid circuits represent promising candidates for viral diagnostics, yet their application in multiplexed viral diagnostics remains challenging. Herein, we present a novel assay for rapid and simultaneous detection of three clinically significant respiratory viruses-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), and influenza A virus (Flu A), using an entropy-driven DNA timer. This innovative strategy combines a gap entropy-driven circuit (EDC) with a toehold-sequestered blocker to temporally gate the cascaded reaction. Precise initiation is achieved through the controlled degradation of the blocker using uracil DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg), orchestrating a time-programmable reaction. Through optimization of blocker and enzyme concentrations, our method achieved precise temporal control over reaction initiation, ensuring high diagnostic accuracy. This assay could detect all three viruses within 45 min in a single reaction, with the limits of detection (LODs) ranging from 4.8 pM to 5.8 pM. Clinical validation using 12 patient samples and three positive quality control samples showed perfect concordance (100%) with RT-PCR results. This work establishes a robust and rapid platform for multiplexed respiratory virus detection and provides a generalizable, time-programmable framework adaptable to other pathogen biosensing applications. Notably, the temporal resolution introduced here operates orthogonally to fluorescence spectral multiplexing, thereby paving the way toward combining temporal and spectral dimensions for higher-order multiplexed detection in a single reaction.