Using chikungunya virus (CHIKV) as a model target, the research team developed a multisite bridging-mediated lateral flow immunoassay (mbLFIA) that enables sensitive and portable detection of mosquito-borne viruses. By redesigning the nucleic acid amplification product to create multiple cross-linking sites and combining this strategy with gold@platinum nanoparticle-based colorimetric enhancement, the method achieved visual detection limits as low as 2 pmol·L−1 for CHIKV. This study provides a promising diagnostic platform for rapid field testing, outbreak surveillance, and infection control, especially in areas where traditional laboratory equipment is difficult to access and resources are limited.
Mosquito-borne viruses such as CHIKV, dengue virus, Zika virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, and Getavirus continue to pose a growing threat to public health as international travel, trade, and the spread of climate-related vectors increase the risk of infection. Nucleic acid tests provide high accuracy because they detect virus-specific genetic sequences and help distinguish between virus variants. However, widely 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 rely on enzymes, thermocycling, fluorescent readers, or other specialized equipment. Existing catalytic hairpin assembly-based lateral flow assays have improved portability but are limited in sensitivity due to the small number of sites available to cross-link colorimetric probes to the test line. These limitations highlight the need for simpler, more sensitive, enzyme-free platforms suitable for virus detection in the field.
The study (DOI: 10.48130/targetome-0026-0016) target tome On April 30, 2026, Yanmin Ju’s team at China Pharmaceutical University reported an enzyme-free mbLFIA strategy that enhances test strip signal through multi-site molecular cross-linking and Au@Pt nanoparticle-catalyzed color deposition.
The researchers first designed a two-round catalytic hairpin assembly (CHA) system containing four hairpin probes: H1, H2, H3, and H4. The presence of CHIKV target RNA causes hybridization between H1 and H2, releasing the target and initiating additional amplification cycles. The resulting H1H2 complex activates H3 and H4 to generate H3H4 hybridization products. Unlike traditional products with limited bridging sites, the H3H4 product was designed with multiple equivalent binding sites, allowing the Au@Pt-DNA probe to be connected to the test line through two bridging mechanisms. This design significantly increased the colorimetric signal. At low product concentrations, the multisite structure produced 10.8- and 9.6-fold stronger signals than the two limited-site design. The team then synthesized Au@Pt nanoparticles and confirmed their structure and composition using transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and related analyses, demonstrating their strong peroxidase-like catalytic activity. These nanoparticles catalyzed the oxidation of 3-amino-9-ethylcarbazole (AEC) and formed an insoluble reddish-brown precipitate on the test line, further amplifying the visual readout. After optimizing the reaction temperature, hairpin ratio, reaction time, probe volume, AEC concentration, hydrogen peroxide concentration, and enhancement time, the assay showed a visual detection range of 2 to 104 pmol.L-1 after colorimetric enhancement compared to 20 to 104 pmol.L-1 for the generic assay. Specificity testing showed that the CHIKV signal was significantly stronger than the signals from ZIKV, DENV, WNV, YFV, JEV, and GETV. For spiked serum, saliva, and urine matrices, recoveries remained within 80% to 120%, indicating good tolerance to biological samples. Finally, mbLFIA identified 16 positives and 20 negatives in 36 mouse serum samples suspected of CHIKV, showing 100% concordance, sensitivity, and specificity with RT-PCR results.
This study provides a new strategy to enhance lateral flow assay signals by increasing the number and efficiency of molecular cross-linking events. This platform combines enzyme-free CHA amplification, multisite hybridization products, and nanozyme-assisted AEC deposition to improve visual sensitivity without the need for complex equipment. The researchers suggest that mbLFIA could support rapid detection of mosquito-borne viruses in clinics, ports, field bases, and low-resource areas, and could be adapted for broader nucleic acid testing applications in infectious disease surveillance.
sauce:
Chinese Academy of Sciences
Reference magazines:
https://doi.org/10.48130/targetome-0026-0016
