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

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.

Keywords: Electrochemical biosensor; Lateral flow assay; Methylene blue; Nucleic acid detection; Quantitative DNA detection.

Fig. 1

(A) MebiQue-LFA: Methylene blue integrated Quantitative electrochemical-Lateral Flow Assay. Schematic overview of the fully integrated device and its connectivity to mobile devices for real-time data transmission. (B) Individual components of the assay: reagent-integrated LFS, cartridge, electrode, and potentiostat for targeted DNA analysis. (C) Schematic depiction of assay mechanism: visual representation of the interaction between target DNA and complementary conjugate pad, forming a duplex, followed by methylene blue intercalation and capture by a dedicated probe in the test line region.

Fig. 2

(A) CAD illustration of the cartridge design portrayed using Fusion360 CAD modeling software. The electrode and LFS placement on the bottom casing are strategically showcased, emphasizing their precise positioning for optimal functionality. The upper casing contains openings allowing the analyte to be introduced to the strip and observe the test line. A graphic overview of the LFS, illustrating the individual pad’s location. (B) Sensitivity test of the LFS, showing performance across the comprehensive range of target concentrations from 0 nM to 1000 nM, and a gradient of blue color can be seen over these concentrations.

Fig. 3

(A) Square wave voltammetry (SWV) measurements of the assay across a spectrum of concentrations (0 nM, 0.001nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM). Each concentration is mapped on the potential vs. current graph, showcasing distinct peaks corresponding to the electrochemical responses. Inset: SWV measurements of the assay for 0 and 0.1 nM. (B) Average current vs. log concentration plot. Each concentration point is plotted against its corresponding average current, and error bars represent the standard deviation of three electrodes.

Fig. 4

(A) Comprehensive analysis of the specificity of the assay using SWV. The graph illustrates the average current vs. potential for 1 µM of mismatches 1 (1 MM), 2 (2 MM), and 3 (3 MM) bp, non-complementary (NC) sequence compared with the 0.1 nM target concentration, and a negative control (0 target concentration). (B) Visual representation of the colorimetric lateral flow strip’s performance after specificity testing, focusing on mismatches at positions 1, 2, and 3 and a non-complementary sequence. (C) ImageJ analysis of the white light intensity of the test region. All the tests were performed for n = 3 devices.

Fig. 5

(A) Process flow of validating the MebiQue-LFA using biological samples. (B) Bar chart illustrating the electrochemical measurement of the MebiQue-LFA in different biological fluids: serum, plasma, whole blood. (C) Calibration curve for detecting ss DNA in 50% diluted blood. The background signal represents 3×standard deviations of the blank (50% diluted blood). Four and Six devices were tested for each measurement in 4B and 4 C respectively.