A multisite bridging mediated lateral flow immunoassay for the enhanced detection of mosquito-borne viruses

The increasing number of infections of mosquito-borne viruses, such as Chikungunya virus (CHIKV), highlight the urgent need for portable diagnostic tools in resource-limited areas, as traditional nucleic acid detection methods are unsuitable for on-site testing because of their reliance on bioenzymes and instruments. Bioenzyme-free nucleic acid amplification methods such as the catalytic hairpin assembly (CHA)-based lateral flow immunoassay (LFIA) have emerged as a promising strategy. However, the detection efficiency is constrained by the limited sites of the amplification products for bridging colorimetric probes to the test line. Herein, we report a multisite bridging mediated LFIA (mbLFIA) strategy, designing amplification products that have multiple sites for bridging colorimetric probes through dual mechanisms, thereby increasing the colorimetric signal by up to 10.8 times at low product concentrations. To further enhance the signal intensity, gold@platinum nanoparticles (Au@Pt NPs) featuring high peroxidase-like activity were used as colorimetric probes, as these can catalyze the oxidation and deposition of 3-amino-9-ethylcarbazole (AEC) on the test line, achieving a detection limit as low as 2 pmol·L⁻¹ for CHIKV. In a clinical evaluation using 36 suspected CHIKV mice serum samples, the method identified 16 positives, with 100% concordance to the reverse transcription polymerase chain reaction (RT-PCR) results, demonstrating high specificity and accuracy. This work provides a novel perspective for optimizing colorimetric signals in LFIA and presents a portable tool for the on-site nucleic acid detection of mosquito-borne viruses.