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

Abstract

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.

Acknowledgments

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).

Ethical statements

This study has been reviewed by the Experimental Animal Ethics Committee of the Institute of Military Veterinary Medicine and complies with animal welfare ethical standards. The ethical review number is IACUC of AMMS-11-2025-024.

Author contributions

The authors confirm their contributions to the work as follows: conceptualization, methodology, software, formal analysis, investigation, data curation, and writing of the original draft: Nie Y; formal analysis and investigation: Shang C, Fan S; methodology, formal analysis, investigation, and writing: Tan X, Song J; methodology: Li W, Hu Q; supervision and funding acquisition: Ju Y; conceptualization, methodology, formal analysis, and investigation: Dai J, Wang Z; review and editing, and funding acquisition: Ju Y. All authors reviewed the results and approved the final version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest

The authors declare that they have no conflict of interest.

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