High throughput influenza A virus detection by isothermal amplification in sequential-injection paper-based microfluidics

Abstract

The recognized impact of epidemics and pandemics caused by Influenza A virus highlights the need for rapid, sensitive, and affordable diagnostic methods. In this work, we propose a molecular detection strategy for Influenza A viruses that combines Electrochemical reverse transcription Loop-Mediated Isothermal Amplification (E-RT-LAMP) using methylene blue (MB) as a redox-active probe, with detection carried out on a sequential-injection paper-based microfluidics (μPAD). The high amplification efficiency of the LAMP technique, following specific target recognition, combined with the intercalation of MB into double-stranded DNA enabled label-free detection of the target sequence through current variation with μPAD. The microfluidic platform was based on the combination of a filter paper disc with 3D pen-templated electrodes, enabling low-cost, portable, and reproducible analysis. The μPAD system exhibited a limit of detection of 9.24 × 101 copies per μL, and following the amplification reaction, detection provided results within seconds (∼3 diagnoses per minute). When tested on a panel of sequenced clinical samples, the assay showed no cross-reactivity with other similar respiratory viruses and demonstrated 100 % accuracy relative to reverse transcription quantitative PCR (RT-qPCR). These results demonstrate the potential of this strategy for point-of-care (POC) diagnostics, offering a promising alternative to conventional laboratory-based molecular methods.