Toward A Modular Label-Free Electrochemical Biosensing Platform Based on a One-Pot Avidin Functionalization of Reduced Graphene Oxide

Abstract

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 sp2 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 x 10-9 M to 2.67 x 10-6 M (R2 = 0.987) and detection limit of 4.4 x 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