Dual-label vertical flow-based electrochemical immunosensor for rapid and simultaneous detection of hepatitis B surface and e virus antigens

The detection of hepatitis B virus (HBV)-associated biomarkers, especially hepatitis B surface (HBsAg) and hepatitis B e (HBeAg) antigens, is of critical significance for the early diagnosis and monitoring of disease progression. However, the detection of these biomarkers has seldom been reported. Herein, we first present a dual-label electrochemical vertical flow-based immunosensor (DL-eVFIS) for the simultaneous detection of HBsAg and HBeAg. DL-eVFIS was engineered to integrate multiple time-sequenced detection procedures into a single device, thus considerably shortening the overall assaying time to 6 min. To enable simultaneous determination, thionine (Th) and ferrocenecarboxylic acid (Fc) were employed as signal tags, as they demonstrate nonoverlapping specific reduction peaks. The appearance of target antigens resulted in a substantial increase in the electrochemical signal response of the redox tags. While requiring only a miniscule sample volume (5 mu L), superior performance was achieved in terms of their limits of detection (LODs) at 1.66 pg/mL for HBsAg and 5.32 pg/mL for HBeAg. DL-eVFIS further demonstrates excellent specificity and selectivity in clinical sample analysis compared with the conventional method. This study contributes to next-generation point-of-care (POC) testing devices for HBV diagnosis and a wide range of biosensing applications.