Highly atom-economic strip for respiratory syncytial virus detection using an entropy-driven circuit-based one-stone-two-birds strategy

Abstract

Lateral flow assays (LFAs) are rapid, inexpensive, and widely used for point-of-care testing (POCT) of viral RNA. However, traditional capture probes, which bind only one type of reporter tag in a single sensing zone, limit amplification efficiency and compatibility with diverse amplification methods. We developed a multi-signal, reverse-transcription-free lateral flow assay for detecting respiratory syncytial virus (RSV) RNA fragments. This assay leverages a chimeric tetrahedral probe combined with an entropy-driven, 'one-stone-two-birds' strategy for enhanced signal amplification. The tetrahedral chimera carries two sorts of capture probes that recognize and capture different signal strand inputs. Entropy-driven circuits (EDC) generate abundant waste chains via a self-feedback cascade reaction, including two biotin-labeled single strands and one duplex. As reporter tags, the two biotinylated single-stranded chains can be simultaneously matched and captured by tetrahedral chimera during one time of sample flow, bridging horseradish peroxidase-coupled streptavidin-modified gold nanoparticles onto the strip to induce coloration. In this process, every EDC cycle can achieve dual signal outputs, akin to "killing two birds with one stone" in the atom-economic signal amplification. Furthermore, the gold nanoparticles carry amounts of horseradish peroxidase, serving as catalytic amplified labels to enhance the colorimetric signal. The strip achieves a broad linear dynamic range and a detection limit of 0.1fM with high specificity for RSV detection. Thus, this lateral flow assay realizes a high atom economy in amplification reaction and offers a rapid, reliable, sensitive, and widely accessible tool for on-site visual detection of viral nucleic acids.