Electroactive reduced graphene oxide for highly sensitive detection of secretory non-structural 1 protein: A potential diagnostic biomarker for Japanese encephalitis virus

Fluorine Doped Tin Oxide (FTO) electrode was fabricated with reduced Graphene Oxide (rGO) for sensitive detection of Japanese encephalitis virus (JEV) non-structural 1 (NS1) protein. Beforehand, in-silico 3D structure, stability, and docking of recombinant JEV NS1 antigen (NS1-Ag) and antibody (Ab) was evaluated. The recombinant NS1 Ag of 42 kDa was produced in-house by successful cloning into pET-28a(+) plasmid and further expressed using BL21 Escherichia coli (E. coli) cells. The NS1 Ag was used to raise polyclonal antibodies (Ab) and both were characterized via Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western Blot, Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF), and Enzyme-Linked Immunosorbent Assay (ELISA). Further characterisation of all binding events such as rGO synthesis, and its conjugation with NS1 Ab, and NS1 Ag were confirmed through Fourier-Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Energy Dispersive X-Ray Analysis (EDX), Scanning Electron Microscopy (SEM), Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The fabricated FTO electrode was optimised for various parameters such as pH, response time, temperature, concentration, and scan rate. The detection of JEV NS1 Ag was performed in buffer (LOD- 0.92 fM) as well in spiked serum (LOD- 1.3 fM) samples. The JEV NS1 Ab showed negligible cross-reactivity with other flaviviral NS1 Ag, provided a rapid response within 5 s, and remained stable up to 4 weeks. Furthermore, the fabricated immunosensor may be a potential candidate for further miniaturisation for accurate and early diagnosis of JEV in clinical samples.