Fluorescein-switching-based lateral flow assay for the detection of microRNAs†

Lateral flow assays (LFAs) are a cost-effective and rapid colorimetric technology that can be effectively used for nucleic acid tests (NATs) in various fields such as medical diagnostics and biotechnology. Given their importance, developing more diverse LFAs that operate through novel working mechanisms is essential for designing highly selective and sensitive NATs and providing insights for designing various practical point-of-care testing (POCT) systems. Herein we report a new type of lateral flow assay (LFA) based on fluorescein-switching, enabled by nucleic acid-templated photooxidation of reduced fluorescein by riboflavin tetraacetate (RFTA). The LFA design leverages the fact that a reduced form of fluorescein, which weakly binds to gold nanoparticle (GNP)-conjugated anti-fluorescein antibodies, is oxidized in the presence of target nucleic acids to yield its native state, which then strongly binds to the antibodies. The study involved designing and optimizing probe sequences to detect miR-6090 and miR-141, which are significant markers for prostate cancer. To minimize background signals of LFAs, sodium borohydride (NaBH₄) was specifically introduced as a reducing agent, and detailed procedures were established. The developed LFA system accurately identified low fmol levels of target microRNAs with minimal false positives, all detectable with the naked eye, making the system a promising tool for point-of-care diagnostics.