de Stigter_2026_Angew.Chem.Int.Ed.Engl__e6796450

Integrated nucleic acid amplification and detection methods that can be conducted at the point of care are critical to advancing infectious disease diagnostics. We previously developed luciferase-intercalating dye conjugates ("LUMIDs") that allow for direct detection of double-stranded DNA (dsDNA) through a simple, ratiometric bioluminescent readout. However, their limited thermostability hampers integration with isothermal nucleic acid amplification methods that provide the specificity and sensitivity required for diagnostics. In this work, we re-engineered LUMID to allow for one-pot integration with loop-mediated isothermal amplification (LAMP) and real-time monitoring of DNA amplification. Luciferase activity and DNA binding were retained at LAMP temperatures (60 degreesC-65 degreesC) by employing a thermostable NanoLuc luciferase variant and an additional thermostable DNA binding protein. To enable continuous monitoring, we additionally introduced a caged luciferin substrate with esterase-controlled release kinetics for sustained light output. These combined advances enabled one-pot LAMP-LUMID assays to detect cancer-associated human papillomavirus (HPV) subtypes with attomolar sensitivity and within - 30 min. Using a simple digital camera as readout, LAMP-LUMID demonstrated robust assay performance in patient samples, showcasing its potential as a rapid and sensitive platform for molecular diagnostics that is particularly attractive for low-resource settings.