Fluorescence/Colorimetric Lateral Flow Immunoassay Based on Dual-Aptamer Domain Recognition and Upconversion Signal Amplification for Sensitive H1N1 Hemagglutinin Detection

Rapid and specific detection of influenza A viruses is critical for pandemic response. However, traditional lateral flow immunoassays (LFIAs) face challenges in sensitivity, signal reliability, and cross-reactivity. Herein, a dual-aptamer-functionalized lateral flow platform is designed, integrating upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) to mitigate these bottlenecks through a synergistic mechanism: near-infrared-excited luminescence resonance energy transfer (LRET) for fluorescence modulation, dual-aptamer domain recognition for specific binding, and fluorescence-colorimetric dual readouts for orthogonal validation. Specifically, LRET serves as the core signal transduction mechanism, with aptamer1-conjugated UCNPs as donors and aptamer2-conjugated AuNPs as acceptors, regulated by target-induced proximity. Dual-aptamers recognizing distinct hemagglutinin (HA) epitopes enhance specificity, minimizing cross-reactivity with non-target subtypes (H5N1, H6N1, etc.) while ensuring high-affinity H1N1 HA binding. Such synergy enables significant signal amplification, achieving a fluorescent limit of detection (LOD) of 2.814 ng mL −1 (2.84–5.33-fold better than traditional AuNP-LFIAs) with a linear range of 4–10 ng mL −1 . Importantly, the dual-mode design integrates upconversion fluorescence and AuNPs colorimetry, mitigating variability, ensuring precision, and enabling 20-minute qualitative detection (up to 100 ng mL −1 ) for point-of-care testing (POCT). Thus, this work presents a scalable POCT paradigm via apt-LRET integration, leveraging dual-aptamer recognition and LRET-mediated amplification for on-site viral diagnostics with pandemic-response timeliness.