Peroxyoxalate chemiexcitation enables optical-excitation-free duplex end-point readout of fluorogenic nucleic acid assays.

Nucleic acid amplification tests (NAATs), including PCR, are considered the gold standard for pathogen detection. Amplification is commonly detected by fluorescence from probes or intercalating dyes, with internally-quenched probes most widely used for enhanced specificity. However, these methods require elaborate instrumentation for fluorescence detection, limiting their usability at point-of-care. We found that the chemical excitation of fluorophores, as used in consumer glow sticks, can be used to detect PCR probe hydrolysis with minimal instrumentation using a smartphone or simple digital camera. By systematically optimizing reagent composition, including hydrogen peroxide, catalyst, viscosifier, and surfactant concentrations, we tuned the reaction kinetics to maximize signal intensity and stability for "Glow" readout. In spiked nasal-swab matrix, smartphone Glow readout detected reactions containing 50 Flu A copies/reaction and 10 SARS-CoV-2 copies/reaction in single-target assays; duplex detection was demonstrated at 500 copies/reaction per target. The assay is compatible with nasal swab extracts and demonstrated two-color duplex readout because the same peroxyoxalate chemistry can chemiexcite multiple fluorophores simultaneously. We establish a general chemiexcitation-based end-point readout architecture for fluorogenic nucleic acid assays, identify fluorophore properties associated with probe-compatible Glow performance in biphasic peroxyoxalate media, and show that this chemistry enables multiplexed post-amplification readout without optical excitation hardware. Peroxyoxalate chemiexcitation may provide a route toward lower-cost molecular diagnostic readout for low-resource settings.