Details

Autor(en) / Beteiligte

• Roberts, Akanksha
• Kesarwani, Veerbhan
• Gupta, Rupal
• Gandhi, Sonu

Titel

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

Ist Teil von

• Biosensors & bioelectronics, 2022-02, Vol.198, p.113837-113837, Article 113837

Ort / Verlag

England: Elsevier B.V

Erscheinungsjahr

2022

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. Expression of recombinant JEV NS1 protein by cloning JEV NS1 insert into pET-28a(+) plasmid. Generation of JEV NS1 Ab by immunization of JEV NS1 purified protein followed by Ab purification and immunoassay development. Development of FTO/rGO/JEVNS1Ab electrode for the detection Ag in spiked serum samples using electrochemical method. [Display omitted] •JEV-NS1 protein expressed in-house to generate polyclonal antibodies assessed by in-silico methods and validated by ELISA.•One-step synthesised rGO and JEV-NS1-antibody used to fabricate the electrode for detection of JEV-NS1 protein biomarker.•The fabricated electrode displayed detection limit of 0.92 fM in buffer and 1.3 fM in spiked serum.•The electrode showed negligible cross reactivity with other flaviviral proteins with storage stability of 4 weeks.