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

TL;DR

This paper introduces a new lateral flow assay using methylene blue to detect nucleic acids with high sensitivity and specificity.

Contribution

The novel use of methylene blue as a colorimetric and electrochemical tracer in nucleic acid detection is introduced.

Findings

MebiQue-LFA achieved a detection limit of 0.62 fM in buffer and 43 fM in diluted blood.

The platform showed high specificity and worked well in complex biological matrices like plasma and serum.

The assay is cost-effective, rapid, and compatible with mobile readout platforms.

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.

The online version contains supplementary material available at 10.1038/s41598-025-19701-4.

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