Influenza virus detection using an electrochemical biosensor based on DSN and RCA

Infectious diseases caused by the influenza virus are the primary global concern. This study aimed to develop an electrochemical biosensor for detecting influenza A H1N1 virus based on duplex-specific nuclease (DSN) and rolling circle amplification (RCA) technologies. A single-stranded DNA (ssDNA) probe, comprising a recognition sequence for a target sequence of influenza virus H1N1-specific RNA sequence and primer sequence for initiating RCA, was designed and synthesized. A specific sequence of target H1N1 RNA triggered cyclical DSN cleavage of the probe. The released primer sequence then initiated RCA to generate detectable ssDNA-labeled multiple biotins. ssDNA was captured by streptavidin (SA) on the electrode surface and labeled with free Streptavidin-Horseradish Peroxidase (SA-HRP). The H1N1 influenza virus was quantitatively analyzed based on the changing current signal generated on the electrode surface. A linear range from 10 pM to 100 nM and a limit of detection of 0.44 pM were recorded. In addition, the biosensor demonstrated excellent selectivity, repeatability, and stability for analyzing H1N1 in real samples. This biosensor could be applied for the clinical diagnosis of influenza A H1N1 virus.