Epitope-level insights for immuno-recognition of JEV NS1 via gold nanoflower- MXene composite.

Early and accurate identification of Japanese encephalitis virus (JEV) is vital for effective outbreak control and surveillance. An electrochemical immunosensor was developed for the detection of JEV non-structural protein 1 (NS1). The JEV NS1 antigen was expressed, purified and subsequently refolded in vitro to recover soluble, functional protein. High-affinity monoclonal antibodies (mAbs) were generated through hybridoma technology. To gain molecular-level insights, antibody gene sequences were decoded using next-generation sequencing (NGS), while molecular docking analysis enabled predictive epitope-paratope mapping. To maximize sensitivity, screen-printed carbon electrodes (SPCEs) were modified with a gold nanoflower/MXene (GNF/MXene) nanocomposite, enhancing the electroactive surface area, and electrical conductivity of the sensor. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) were employed to analyse the electrochemical reactions. The sensor exhibited a low limit of detection (LOD) of 0.35 fM across a broad range concentration range of JEV NS1 (1 fM-1 μM). The immunosensor was further evaluated with clinical samples and sensor showed high specificity (100%), sensitivity (95%), and accuracy (96.66%) with no cross-reactivity to other flavivirus NS1 antigens, with a rapid response of 10 min and maintained excellent stability over 4 weeks.