Subtyping of influenza A H1N1 virus using a label-free electrochemical biosensor based on the DNA aptamer targeting the stem region of HA protein

Highlights: • An ssDNA aptamer selected for subtyping of influenza A H1N1 viruses via SELEX is reported for the first time. • Aptamers that recognize H1N1 subtypes alone were selected using mini HA protein and whole H1N1 viruses. • The dissociation constant of 19.2 nM was obtained for aptamer V46. • The electrochemical sensor based on the DNA aptamer distinguished H1N1 subtypes from other subtypes of influenza A virus. -- Abstract: Rapid subtyping of influenza viruses in clinical laboratories has been increasingly important because three subtypes (seasonal H1N1, H3N2, and 2009 H1N1) of influenza A virus currently disseminated in humans have variable susceptibilities to antiviral drug. Herein, we present DNA aptamers for selective detection of influenza A H1N1 (seasonal and 2009 pandemic H1N1) viruses by targeting recombinant influenza A mini-hemagglutinin (mini-HA) protein (the stable stem region of HA) and whole H1N1 viruses. The dissociation constants (K_D) of aptamer candidates V46 and V57 were 19.2 nM and 29.6 nM, respectively, according to electrochemical characterization (differential pulse voltammetry), demonstrating strong binding to mini-HA. In comparison, the K_D of the influenza virus antibodies is in the range of 1 μM–10 nM. Aptamer V46 showed higher specificity and binding affinity to the mini-HA protein and H1N1 subtypes, and it was also incorporated into an indium tin oxide-based electrochemical sensor, showing sensitive and specific detection of H1N1 viruses, with a limit of detection (LOD) of 3.7 plaque-forming units per mL. The binding affinity, specificity, and LOD achieved with the electrochemical sensor suggest that it can be used for rapid subtyping of H1N1. We also propose that this aptamer can be used for the neutralization of H1N1 subtypes, suggesting potential therapeutic and diagnostic applications.