Engineering a surfactant-free Au@nitrogen-doped graphene quantum dot heterointerface for ultrasensitive electrochemical detection of miR-9

Sensitive and reliable electrochemical detection of circulating microRNAs requires sensing interfaces that combine rapid electron transfer, stable probe immobilization, and high analytical performance in complex biological media. Herein, a label-free electrochemical biosensor based on an in situ engineered, surfactant-free Au@nitrogen-doped graphene quantum dot (Au@N-GQD) heterointerface was developed for the sensitive detection of microRNA-9 (miR-9), a clinically relevant biomarker associated with lung cancer progression. The sensing interface was constructed by sequential modification of a fluorine-doped tin oxide (FTO) electrode with nitrogen-doped graphene quantum dots, followed by electrodeposition of gold nanostructures and immobilization of a thiolated DNA capture probe. The synergistic integration of Au nanostructures and N-GQDs provided a highly conductive heterointerface, facilitating electron-transfer kinetics and probe immobilization through Au-S interactions without the need for external labels or enzymatic amplification. The successful fabrication of the sensing interface was confirmed by complementary spectroscopic, microscopic, and electrochemical characterization. Electrochemical measurements using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS), demonstrated a limit of detection of 0.018 aM over a broad investigated concentration range extending from zeptomolar to micromolar levels. The biosensor exhibited high selectivity toward miR-9 in the presence of interfering microRNAs, including miR-21 and miR-223, and enabled reliable detection in spiked human serum samples, demonstrating its potential applicability in complex biological matrices. Overall, the proposed sensing strategy provides a robust, and reproducible platform for sensitive microRNA analysis and shows promise for future applications in lung cancer biomarker detection and point-of-care diagnostics.