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

Abstract 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.