Preparation of an ultrasensitive electrochemical immunosensor for the rapid detection of 19-nortestosterone based on polyvinyl alcohol/polyacrylic acid electrospun nanofiber mat

19-nortestosterone (19-NT), being regarded as the residual marker of banned synthetic steroid hormone nandrolone phenylpropionate, could cause adverse effects when consumed by human. To ensure food safety and minimize 19-NT toxicity, herein, an innovative electrochemical immunosensor for detection of 19-NT was constructed based on the modified polyvinyl alcohol/polyacrylic acid (PVA/PAA) nanofiber mat (NFM). The detection was achieved via a direct competition for binding to horse radish peroxidase-tagged anti-19-NT nanobody between free 19-NT and the 19-NT-ovalbumin covalently immobilized onto the NFM, and the amperometric response of immunosensor was analyzed using cyclic voltammetry. Under the optimal preparation conditions, the PVA/PAA NFM with appropriate hydrophilicity and ultra-high surface was obtained and characterized, and its electrochemical performance exhibited a 20-times increased response compared to the PVA/PAA casting membrane. The established immunosensor possessed high sensitivity towards 19-NT with a detection limit of 2.14 pg/mL, a 46-fold times lower compared to the traditional ic-ELISA of pure anti-19-NT nanobody. Recovery rates from the spiked animal urine samples ranged from 97.09 % to 117.32 %. Moreover, the sensor showed good reproducibility and stability over 6 weeks, suggesting that the nanofiber-based biosensor is a promising alternative for the fast and sensitive determination of 19-NT.