An ultrasensitive sandwich-type electrochemical immunosensor for HBsAg detection based on Au/PANI interface and microenvironment-regulated nanozyme

The timely and precise detection of hepatitis B surface antigen (HBsAg) remains essential for the clinical screening and therapeutic monitoring of hepatitis B virus infection. Electrochemical immunosensors have emerged as promising tools owing to their high sensitivity, rapid response, and potential for point-of-care testing. However, ultrasensitive detection is still constrained by the intrinsic “pH mismatch” of conventional nanozymes, whose catalytic activity is significantly suppressed under near-neutral conditions required for stable immunoreactions. Herein, an ultrasensitive sandwich-type electrochemical immunosensor was engineered for HBsAg detection by integrating conductive interface engineering with microenvironment-regulated catalytic amplification. Specifically, a highly conductive Au/polyaniline nanocomposite was assembled on a glassy carbon electrode to increase the electroactive surface area. Stable immobilization of capture antibodies was achieved through Au–NH₂ interactions, thereby promoting interfacial charge-transfer efficiency. To maximize signal amplification, a multifunctional PCN-222-Fe@PAA@PtCu nanoplatform was developed as a robust signal probe. The in-situ integration of PAA provided a proton reservoir to maintain nanozyme catalytic activity under neutral conditions, while PtCu nanodendrites introduced synergistic active sites, significantly enhancing the sensitivity of the H₂O₂-based electrochemical response. The sensor exhibited a wide linear range of 0.0005–10 ng/mL and a low detection limit of 135 fg/mL. Its reliability was further validated by excellent specificity and stability in clinical serum samples. This work provides an effective strategy to overcome the pH limitation of nanozyme-based immunosensors and offers a promising platform for ultrasensitive HBsAg detection in clinical diagnostics.