A colorimetric/electrochemical dual-mode aptasensor for Golgi protein 73 detection using TMB as a bifunctional probe

Golgi protein 73 (GP73) has emerged as a critical biomarker for the diagnosis of hepatocellular carcinoma (HCC), and its detection is essential for the effective treatment of this disease. In this study, we developed a colorimetric/electrochemical dual-mode aptasensor for GP73 detection using a hemin-reduced graphene oxide‑manganese oxide (H-rGO-Mn₃O₄) nanozyme and the bifunctional probe, 3,3',5,5'-tetramethylbenzidine (TMB). The GP73-specific aptamer (Apt) was conjugated to the H-rGO-Mn₃O₄ nanozyme, which was then immobilized on the surface of a gold nanoparticle-modified screen-printed electrode (Au NPs/SPE) through hybridization with a complementary DNA strand (cDNA) attached to the electrode. TMB served as a dual-signal probe for both colorimetric and electrochemical detection. In the presence of GP73, Apt binds specifically to the target, forming a H-rGO-Mn₃O₄-Apt/GP73 complex structure. This binding event causes the nanozymes to detach from the electrode surface, reducing its peroxidase activity while simultaneously restoring the electrochemical signal. Under optimized conditions, the dual-mode aptasensor exhibited excellent linear responses for GP73 concentrations ranging from 0.0001 to 100 ng/mL, with limits of detection (LOD) of 0.1 pg/mL for both the colorimetric and electrochemical modes. The aptasensor was successfully applied to measure GP73 levels in serum samples, yielding highly consistent results. Its performance was further validated by comparison with the standard ELISA method. Compared to single-signal aptasensors, this dual-mode approach not only improves detection sensitivity but also minimizes the risk of false positives or false negatives, making it a more reliable tool for accurate GP73 detection in clinical diagnostics.