A research team has developed a multisite-mediated lateral flow immunoassay (mbLFIA) that enables highly sensitive, portable detection of mosquito-borne viruses, using Chikungunya virus (CHIKV) as a model target. By redesigning nucleic acid amplification products to create multiple bridging sites and coupling this strategy with gold@platinum nanoparticle-based colorimetric amplification, the method achieved an optical detection limit as low as 2 pmol·L−1 for CHIKV. The work offers a promising diagnostic platform for rapid field testing, outbreak surveillance and infection control, especially in resource-constrained areas where conventional laboratory instruments are difficult to access.
Mosquito-borne viruses, such as CHIKV, dengue virus, Zika virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, and Getah virus, continue to pose increasing threats to public health as international travel, trade, and the expansion of climate-related vectors increase the risks of transmission. Nucleic acid testing provides high accuracy because it detects native virus gene sequences and can help distinguish virus variants. However, commonly used methods such as reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification, rolling circle amplification, and CRISPR-based assays often depend on enzymes, thermal cycling, fluorescence readers, or other specialized equipment. Existing lateral flow assays based on a catalytic hairpin arrangement have improved portability, but their sensitivity is limited by the small number of sites available for bridging colorimetric probes in the test line. These limitations highlight the need for a simpler, more sensitive enzyme-free platform suitable for in situ virus detection.
A study (DOI: 10.48130/targetome-0026-0016) published in Target on April 30, 2026 by Yanmin Ju’s group, China Pharmaceutical University, reports an enzyme-free mbLFIA strategy that enhances test strip signals via multisite molecular bridging and Au@Pt nanoparticle-catalyzed dye deposition.
The researchers first designed a two-round catalytic hairpin array (CHA) system that includes four hairpin detectors, H1, H2, H3, and H4. When CHIKV target RNA is present, it activates hybridization between H1 and H2, freeing the target to initiate additional cycles of amplification. The resulting H1H2 complex then activates H3 and H4 to generate H3H4 hybridization products. In contrast to conventional products with limited bridging sites, the H3H4 products were constructed with multiple equivalent binding sites, allowing them to bind Au@Pt-DNA probes to the test line via two bridging mechanisms. This design significantly increased the colorimetric signal: at low product concentration, the multisite structure produced a 10.8-fold and 9.6-fold stronger signal than two site-restricted designs. The team then synthesized Au@Pt nanoparticles, confirmed their structure and composition using transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction and related analyses, and demonstrated their strong peroxidase-like catalytic activity. These nanoparticles catalyze the oxidation of 3-amino-9-ethylcarbazole (AEC), forming an insoluble brown-red precipitate on the test line and further enhancing the optical reading. After optimizing the reaction temperature, hairpin ratios, reaction time, probe volume, AEC concentration, hydrogen peroxide concentration and amplification time, the assay showed an optical detection range from 2 to 104 pmol·L−1 after colorimetric amplification, compared to 20 to 104 pmol·L−1 for the general analysis. Specificity tests showed that the CHIKV signal was significantly stronger than the signals from ZIKV, DENV, WNV, YFV, JEV and GETV. In spiked matrices of serum, saliva and urine, recovery rates remained between 80%-120%, indicating good tolerance in biological samples. Finally, in 36 suspected mouse CHIKV serum samples, mbLFIA detected 16 positive and 20 negative, matching the RT-PCR results with 100% concordance, sensitivity, and specificity.
This study provides a novel strategy to enhance lateral flow analysis signals by increasing the number and efficiency of molecular bridging events. By combining enzyme-free CHA amplification, multisite hybridization products, and nanoenzyme-assisted AEC deposition, the platform improves optical sensitivity without requiring complex instrumentation. The researchers suggest that mbLFIA could support the rapid detection of mosquito-borne viruses in clinics, ports, field stations and low-resource areas, and can be adapted for broader applications of nucleic acid testing in infectious disease surveillance.
