Orthogonal signal-transducing electrochemical dual-aptasensor for ultrasensitive parallel detection of GP73 and AFP in serum

Background Golgi protein 73 (GP73) has emerged as a promising serological biomarker for hepatocellular carcinoma (HCC), with its combination with alpha-fetoprotein (AFP) substantially improving diagnostic precision in early-stage HCC detection. To address this clinical need, we constructed an electrochemical dual-aptasensor for parallel quantification of GP73&AFP. Results GP73 aptamer (GP73 Apt ) functionalized with reduced graphene oxide-ferrocene-polyaniline (rGO-Fc-PANi) nanocomposites and AFP aptamer (AFP Apt ) conjugated with nitrogen-doped reduced graphene oxide-cuprous oxide (NrGO-Cu 2 O) nanostructures were employed as the dual-signal probes. Upon GP73&AFP binding, the GP73 Apt -GP73 interaction induced double-helix dissociation of aptamer-complementary DNA (cDNA) complexes, stabilizing the Fc-mediated electrochemical structure and amplifying Fc redox current. Conversely, AFP Apt -AFP interaction induced steric hindrance through aptamer-protein complex formation, synergistically combined with dielectric property changes to suppress Cu 2 O redox signals. Under optimal conditions, the aptasensor demonstrated logarithmic linear ranges of 0.001–100.0 ng/mL for both biomarkers, achieving lower LOD of 0.48 pg/mL (GP73) and 1.77 pg/mL (AFP). Additionally, GP73&AFP were directly measured in human serum samples and demonstrated excellent reproducibility with RSD of 1.15–3.52 % for GP73 and 3.96–5.97 % for AFP. Significance This work presents a groundbreaking advancement in HCC diagnostics through the development of a dual-mode electrochemical aptasensor. The system's sensitive simultaneous detection capability, combined with operational stability in complex biological matrices, positions it as a transformative tool for early-stage HCC screening and management.