Toward a modular label-free electrochemical biosensing platform based on a one-pot avidin functionalization of reduced graphene oxide

A one-pot, ultrasound-assisted and environmentally responsible strategy is proposed for the simultaneous partial reduction and biofunctionalization of graphene oxide (GO), using L-tyrosine (L-tyr) and ascorbic acid (AA) as mild and complementary reducing agents. This synergistic approach enables controlled partial restoration of the sp 2 carbon framework while preserving oxygen functionalities required for biomolecular anchoring. Avidin is incorporated in situ during the reduction process, eliminating post-synthetic modification steps while enabling its functional role in biotinylated recognition element immobilization. The resulting avidin-functionalized reduced graphene oxide (rGO) provides a functional electrochemical interface for the oriented immobilization of biotinylated recognition elements through the avidin-biotin affinity interaction. This immobilization strategy was conceived to facilitate the assembly of different biosensing interfaces without requiring surface re-engineering. The platform was implemented as a label-free electrochemical interface and characterized by electrochemical impedance spectroscopy to monitor interfacial assembly and biorecognition events. As a proof-of-concept, the proposed immobilization strategy was experimentally validated using the thrombin-binding aptamer as a representative biotinylated recognition element for the detection of human α-thrombin (α-th). The platform exhibited a linear response from 1.34 × 10 −9 M to 2.67 × 10 −6 M ( R 2 = 0.987) and a detection limit of 4.4 × 10 −10 M, demonstrating selective response in the presence of potentially interfering proteins and enriched human serum samples. These results demonstrate a sustainable one-pot strategy for producing avidin-functionalized rGO and establish a proof-of-concept for an avidin-based electrochemical biointerface. Although the present study experimentally validates the approach using a single aptamer-target pair, the proposed immobilization strategy may enable the future development of adaptable electrochemical biosensing platforms employing different biotinylated recognition elements without modifying the underlying transducer.