Thermostable Bioluminescent Intercalating Dyes for Real‐Time, Integrated Nucleic Acid Amplification and Detection

ABSTRACT 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°C–65°C) 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.