Methylene blue as a new signal tracer for nucleic acid-based lateral flow assay

Abstract

Abstract We report the development of MebiQue-LFA, a nucleic acid-integrated lateral flow assay that utilizes methylene blue (MB) as a new colorimetric signal tracer for detecting nucleic acids. MebiQue-LFA advances LFA technology by enabling visual detection via blue dye and enhancing sensitivity through integrated electrochemical transduction on the same platform. The electrochemical readout provides a sensitive and quantifiable alternative when visual detection is limited at low analyte concentrations. To demonstrate dual-mode detection, the assay was designed to target a model single-stranded DNA sequence, achieving a detection limit (DL) of 0.62 fM and 43 fM (~ 100 copies/µL) in buffer and contrived diluted (50%) blood respectively, with a dynamic range spanning 1 pM to 1 µM. The platform exhibited high specificity, discriminating single-base mismatches and non-complementary sequences. Furthermore, MebiQue-LFA demonstrated robust performance in complex biological matrices, including plasma and serum, achieving a DL of 16 pM (~ 105 copies/µL). The platform is cost-effective (~ 3 CAD per test), rapid (15 min), stable in different storage conditions and compatible with mobile readout platforms, highlighting its potential for decentralized and point-of-care nucleic acid diagnostics.