Ultrasensitive electrochemical detection of hepatitis C virus core antigen using terminal deoxynucleotidyl transferase amplification coupled with DNA nanowires

Early diagnosis of hepatitis C virus (HCV) infection is essential to prevent disease from spreading and progression. Herein, a novel electrochemical biosensor was developed for ultrasensitive detection of HCV core antigen (HCVcAg) based on terminal deoxynucleotidyl transferase (TdT) amplification and DNA nanowires (DNW). After sandwich-type antibody-antigen recognition, the antibody-conjugated DNA was pulled to the electrode surface and further extended into a long DNA sequence by robust TdT reaction. Then, large numbers of methylene blue–loaded DNW (MB@DNW) as signal labels are linked to the extended DNA sequence. This results in an amplified electrochemical signal for HCVcAg determination, typically measured at around −0.25 V (Ag/AgCl). Under the optimum conditions, the proposed biosensor achieved a wide linear range for HCVcAg from 0.1 to 312.5 pg/mL with a low limit of detection of 32 fg/mL. The good practicality of the biosensor was demonstrated by recovery experiment (recoveries from 98 to 104% with RSD of 2.5–4.4%) and comparison with enzyme-linked immunosorbent assay (ELISA). Given the highlighted performance, the biosensor is expected to act as a reliable sensing tool for HCVcAg determination in clinics. Graphical abstract Schematic representation of the ultrasensitive electrochemical biosensor based on terminal deoxynucleotidyl transferase (TdT) amplification linked with methylene blue–loaded DNA nanowires (MB@DNW), which can be applied to the determination of hepatitis C virus core antigen (HCVcAg) in clinical samples. dTTPs, 2′-deoxythymidine 5′-triphosphate.