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Updated: Jan 8, 2026
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Self-enhancing triplex lateral flow assay for simultaneous detection of influenza A, influenza B, and SARS-CoV-2
Lingyan Kang1, Tao Xu1, Zhimeng Fan1
1College of Medicine and Biological Information Engineering, Northeastern University, Shenyang, 110169, China.
Insights
A novel self-enhancing lateral flow immunoassay (LFIA) boosts sensitivity for detecting respiratory pathogens like IAV, IBV, and SARS-CoV-2. This equipment-free, one-step diagnostic offers improved detection of weak-positive samples for reliable point-of-care screening.
Area of Science:
- Biomedical Engineering
- Diagnostic Assays
- Nanotechnology
Background:
- Lateral flow immunoassays (LFIA) are crucial for point-of-care diagnostics but often lack sensitivity, especially for detecting low pathogen concentrations.
- Improving LFIA sensitivity without complicating the user workflow is a significant technical challenge.
Purpose of the Study:
- To develop a self-enhancing lateral flow immunoassay (SET-LFIA) that increases analytical sensitivity and maintains a simple, one-step format.
- To enable reliable detection of respiratory pathogens, including influenza A virus (IAV), influenza B virus (IBV), and SARS-CoV-2, with enhanced accuracy for weak-positive samples.
Main Methods:
- A novel SET-LFIA design incorporating two enhancement pads (Au³⁺ and NH₂OH·HCl) positioned beneath the detection layer.
- Sequential reagent release triggered by capillary flow, inducing in situ gold nanoparticle growth post-binding to amplify the signal.
- Optimization of reagent concentrations and pad placement to maximize signal enhancement while preserving immunoreaction integrity.
Main Results:
- The SET-LFIA achieved significantly lower detection limits: 1.0 ng/mL for IAV, 1.0 ng/mL for IBV, and 0.2 ng/mL for SARS-CoV-2.
- Demonstrated 5-25 fold sensitivity gains compared to conventional LFIA, with improved discrimination of weak-positive samples and reduced false negatives.
- Exhibited excellent stability (>85% signal retention after 21 days at 45°C) and robust multiplex detection in clinical samples.
Conclusions:
- The developed SET-LFIA provides a practical and scalable solution for rapid, on-site respiratory pathogen screening.
- The integrated self-enhancement strategy effectively boosts assay sensitivity without additional user steps, addressing a key limitation of current LFIAs.
- This technology holds promise for improving the reliability and accessibility of point-of-care diagnostics for infectious diseases.
Abstract:
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.
