Mosquito‑borne viruses, including chikungunya, dengue, Zika, and Japanese encephalitis viruses, pose a growing threat to public health. Traditional gold‑standard methods such as RT‑PCR require complex equipment and enzymatic amplification, making them unsuitable for rapid point‑of‑care testing in outbreak zones. While enzyme‑free isothermal amplification approaches like catalytic hairpin assembly (CHA) paired with lateral flow immunoassay (LFIA) have shown promise, their sensitivity is severely limited by low bridging efficiency between amplification products and colorimetric probes on the test strip.
The new mbLFIA platform overcomes this bottleneck via two key innovations:
Dual‑mechanism multisite bridging in CHA‑generated hybridization products, boosting colorimetric signal by up to 10.8‑fold at low concentrations.
Gold‑platinum nanoparticles (Au@Pt NPs) with strong peroxidase‑like activity as colorimetric reporters, which catalyze localized deposition of 3‑amino‑9‑ethylcarbazole (AEC) to produce stable, intense red‑brown bands visible to the naked eye.
Under optimized conditions, mbLFIA achieved a visual limit of detection (vLOD) as low as 2 pmol·L⁻¹ for CHIKV, 10× more sensitive than conventional CHA‑LFIA formats. In blinded testing with 36 mouse serum samples from suspected CHIKV infection, the assay correctly identified 16 positives with 100% concordance to RT‑qPCR results, demonstrating perfect clinical sensitivity and specificity (AUC = 1.0). The method also showed high selectivity against other arboviruses including dengue, Zika, yellow fever, West Nile, and Japanese encephalitis viruses.
By redesigning how amplification products bridge detection probes and using nanozyme‑enhanced signal amplification, this research turned a simple test strip into an ultrasensitive diagnostic tool that works without enzymes or expensive machines. This makes mbLFIA particularly valuable for rapid surveillance and early intervention during arbovirus outbreaks in low‑resource regions.
The complete assay workflow includes viral RNA extraction, two‑cycle CHA amplification, strip‑based readout, and AEC signal enhancement, all performed under mild isothermal conditions with minimal equipment. The platform maintains robust performance in biological matrices including serum, urine, and saliva, supporting broad translational potential.
This study establishes multisite bridging as a generalizable strategy to boost colorimetric performance in lateral flow assays and provides a ready‑to‑use, cost‑effective tool for on‑site nucleic acid detection of mosquito‑borne viruses. Future development will focus on integrating microfluidics and multiplexing to enable simultaneous screening of multiple pathogens in a single test.
This work entitled “A multisite bridging mediated lateral flow immunoassay for the enhanced detection of mosquito-borne viruses” was published online April 30, 2026, in Targetome.
DOI：10.48130/targetome-0026-0016