Paper-based enzyme-free immunoassay for rapid detection and subtyping of influenza A H1N1 and H3N2 viruses

• Development of a paper-based enzyme-free sandwich immunoassay.
• Optimization of the conditions of paper-based immunoassay.
• Detecting and subtyping of influenza viruses with the detection limits of 2.7 × 103 pfu/assay for H1 detection and 2.7 × 104 pfu/assay for H3 detection.
• Demonstration of influenza screening with less sample and reagent volume (5 μL) in shorter period of time (around 1 h).
• Evaluation of the paper-based immunoassay using infected cell lysate and clinical samples.

Development of rapid screening in the ambulatory environment is the most pressing needs for the control of spread of infectious disease. Despite there are many methods to detect the immunoassay results, quantitative measurement in rapid disease screening is still a great challenge for point-of-care applications. In this work, based on the internal structural protein, i.e., nucleoprotein (NP), and outer surface glycoproteins, i.e., H1 and H3, of the influenza viruses, specific and sensitive immunoassay on paper-based platform was evaluated and confirmed. Detection and subtyping of influenza A H1N1 and H3N2 viruses found in people were demonstrated by colorimetric paper-based sandwich immunoassay. Concentration-dependent response to influenza viruses was shown and the detection limits could achieve 2.7 × 103 pfu/assay for H1 detection and 2.7 × 104 pfu/assay for H3 detection, which are within the clinical relevant level. Moreover, detection of influenza virus from infected cell lysate and clinical samples was demonstrated to further confirm the reliability of the paper-based immunoassay. The use of paper for the development of diagnostic devices has the advantages of lightweight, ease-of-use, and low cost and paper-based immunoassay is appropriate to apply for rapid screening in point-of-care applications.

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