Enzymatic DNA Reaction Networks for Orchestrating Stimuli‐Dependent Temporal Molecular Pulse

Temporal dynamics are a hallmark of cellular information processing, yet cell-free biosensors still mainly rely on amplitude-based fluorescence readouts that face multiplex limits from limited spectral channels, cross-talk, and rising complexity. Here we present an enzymatic DNA reaction network (EDRN) that introduces time as an orthogonal coding dimension by translating stimuli into programmable temporal pulses. EDRN integrates a polymerase-based concentration converter that normalizes inputs into a standardized universal strand (Us) at set doses with an exonuclease-driven temporal decoder that converts Us dose into transient fluorescence pulses. This modular separation provides orthogonal enzymatic control over pulse amplitude and lifetime, enabling a wide programmable range without delicate structure-dependent fine-tuning. By tuning Us production through single- or double-layer converters, pulse lifetimes can be programmed from ∼10 min to ∼5 h and temporal signatures assigned across targets. As a proof of concept, we demonstrate multiplex bacterial nucleic-acid detection in one tube, where targets are resolved by time-color encoding, achieving ten-plex readout using four fluorophores with multiple temporal windows. Clinical validation on 32 specimens (22 positives and 10 healthy controls) showed consistency with sequencing. These results establish a general stimulus-to-time strategy for nucleic-acid circuits and expand the multiplexing capacity of fluorescence-based cell-free biosensing.