Self-enhancing triplex lateral flow assay for simultaneous detection of influenza A, influenza B, and SARS-CoV-2

Achieving multiplexed, equipment-free, and sensitivity-enhanced antigen detection within a true one-step lateral flow format remains a technical challenge. Lateral flow immunoassays (LFIAs) are widely used for point-of-care diagnostics, yet their sensitivity-particularly in weak-positive specimens-often limits reliable pathogen identification. Herein, we enhance the analytical performance of LFIA by introducing a structurally integrated self-enhancement strategy that maintains the simplicity of one-step operation without requiring additional user steps. In the proposed SET-LFIA, two enhancement pads loaded with Au³⁺ and NH₂OH·HCl are positioned beneath the detection layer and isolated by a double-sided adhesive barrier, enabling sequential reagent release along the capillary flow path and inducing localized in situ gold growth during the post-binding phase. This design strengthens the visual signal while preserving the immunoreaction process. Under optimized conditions, the SET-LFIA achieved detection limits of 1.0 ng/mL for IAV, 1.0 ng/mL for IBV, and 0.2 ng/mL for SARS-CoV-2, corresponding to 5-25-fold sensitivity gains over conventional LFIA. Tests using spiked samples demonstrated markedly improved discrimination of weak-positive specimens and reduced false-negative outcomes. In addition, stability evaluation showed >85 % signal retention after 21 days at 45 °C, and clinical samples further confirmed robust multiplex detection across all three targets. With its enhanced sensitivity, one-step workflow, and compatibility with industrial lamination, the SET-LFIA represents a practical and scalable solution for rapid on-site respiratory pathogen screening.