Sensitive electrochemical detection of vaccinia virus in a solution containing a high concentration of l-ascorbic acid

Abstract

Washing processes cannot fully remove interfering species that remain on biosensing surfaces when a sample solution contains a high concentration of interfering species. This study reports an immunosensing scheme employing electroreduction-based electrochemical–chemical (EC) redox cycling that allows sensitive detection of vaccinia virus (VV) in a solution containing a high concentration of L-ascorbic acid (AA). To obtain high signal amplification, an enzymatic reaction by β-D-galactosidase (Gal) is combined with electroreduction-based EC redox cycling by an oxidant. Among the four possible oxidants (KIO₃, NaClO, Ag₂O, and H₂O₂), KIO₃ shows the highest signal-to-background ratio and is chosen. During an incubation period of 10 min, Gal converts β-D-galactopyranoside into p-aminophenol (AP), which is oxidized to p-quinone imine (QI) by KIO₃. When −0.05 V vs. Ag/AgCl is applied to an immunosensing electrode, QI is reduced to AP, and the regenerated AP is then reoxidized by KIO₃. The electroreduction-based EC redox cycling is induced. An indium−tin oxide electrode modified with reduced graphene oxide and an applied potential of −0.05 V are used to achieve low and reproducible background currents, slow O₂ reduction, and fast electroreduction of QI. KIO₃ favorably converts AA into noninterfering species during the incubation period. The detection limit for VV in commercial 50% mandarin juice (AA concentration = 0.7 mM) is 4 × 10³ plaque-forming unit (PFU) per mL. The new EC redox cycling scheme is promising for sensitive detection of proteins, viruses, and bacteria in solutions containing high concentrations of AA.