Mosquito-borne viruses, including CHIKV, Dengue virus, Zika virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus, and Getah virus, continue to pose growing threats to public health as international travel, trade, and climate-related vector expansion increase transmission risks. Nucleic acid testing provides high accuracy because it detects intrinsic viral gene sequences and can help distinguish viral 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 depend on enzymes, thermal cycling, fluorescence readers, or other specialized equipment. Existing catalytic hairpin assembly-based lateral flow assays have improved portability, but their sensitivity is limited by the small number of sites available for bridging colorimetric probes to the test line. These limitations highlight the need for a simpler, more sensitive, enzyme-free platform suitable for on-site viral detection.
A study (DOI: 10.48130/targetome-0026-0016) published in Targetome on 30 April 2026 by Yanmin Ju's team, China Pharmaceutical University, reports an enzyme-free mbLFIA strategy that strengthens test-strip signals through multisite molecular bridging and Au@Pt nanoparticle-catalyzed color deposition.
The researchers first designed a two-round catalytic hairpin assembly (CHA) system involving four hairpin probes, H1, H2, H3, and H4. When CHIKV target RNA is present, it triggers hybridization between H1 and H2, releasing the target to start additional amplification cycles. The resulting H1H2 complex then activates H3 and H4 to generate H3H4 hybridization products. Unlike conventional products with limited bridging sites, the H3H4 products were engineered with multiple equivalent binding sites, allowing them to connect Au@Pt-DNA probes to the test line through two bridging mechanisms. This design markedly increased the colorimetric signal: at low product concentration, the multisite structure produced a signal 10.8 times and 9.6 times stronger than two limited-site 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 catalyzed the oxidation of 3-amino-9-ethylcarbazole (AEC), forming an insoluble brown-red precipitate on the test line and further amplifying the visual readout. After optimizing reaction temperature, hairpin ratios, reaction time, probe volume, AEC concentration, hydrogen peroxide concentration, and enhancement time, the assay showed a visual detection range from 2 to 10⁴ pmol·L−1 after colorimetric enhancement, compared with 20 to 10⁴ pmol·L−1 for the general assay. Specificity tests showed that the CHIKV signal was significantly stronger than signals from ZIKV, DENV, WNV, YFV, JEV, and GETV. In spiked serum, saliva, and urine matrices, recovery rates remained within 80%–120%, indicating good tolerance to biological samples. Finally, in 36 suspected CHIKV mouse serum samples, mbLFIA identified 16 positives and 20 negatives, matching RT-PCR results with 100% concordance, sensitivity, and specificity.
This study provides a new strategy for strengthening lateral flow assay signals by increasing the number and efficiency of molecular bridging events. By combining enzyme-free CHA amplification, multisite hybridization products, and nanozyme-assisted AEC deposition, the platform improves visual sensitivity without requiring complex instruments. The researchers suggest that mbLFIA could support rapid detection of mosquito-borne viruses in clinics, ports, field stations, and low-resource areas, and may be adapted for broader nucleic acid testing applications in infectious disease surveillance.
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References
DOI
10.48130/targetome-0026-0016
Original Source URL
https://doi.org/10.48130/targetome-0026-0016
Funding information
This study was financially supported by the National Natural Science Foundation of China (22574173), the Scientific Research Program Project of Drug Regulatory Science, Jiangsu Provincial Medical Products Administration, China (202518), and the Project Program of State Key Laboratory of Natural Medicines (China Pharmaceutical University) (SKLNMZZ2024JS46, SKLNMZZ202510).
About Targetome
Targetome refers to the complete collection of molecular targets (e.g., proteins, RNA or DNA) that interact with and mediate the effect of a specific biomolecule, such as a drug, toxin, metabolites, transcription factor or microRNA, within a biological system. Targetome is an open access journal publishing rigorously peer-reviewed original research articles, reviews, break-through methods, and perspectives that advance our understanding, identification and validation of molecular targets for new drug development.