A Novel Label-Free EC Aptasensor for Early Detection of H9N2 Influenza Using FFT-square wave voltammetry and Validated by Molecular Docking

Abstract

H9N2 avian influenza threatens global poultry production and human health through cross-species transmission and its role as a genetic reservoir for emerging influenza strains. This dual risk underscores the urgent need for rapid and reliable early detection. Here we report the first biosensor deployment of the B4 anti-H9N2 hemagglutinin (HA) aptamer as the recognition element for electrochemical detection, translating its previously reported HA affinity into a practical sensing platform and further supporting this affinity through molecular docking analysis. In this study, the sensing interface integrates cerium oxide nanoparticles (CeO₂) and electrochemically reduced graphene oxide (rGO) with electrodeposited gold nanoparticles (AuNPs) on a gold electrode, forming a conductive and stable surface that enables oriented immobilization of a PolyA-PolyT-modified aptamer via adenine-gold affinity. Fast Fourier Transform square-wave voltammetry (FFT-SWV) was employed for signal transduction, enabling frequency-domain extraction of Faradaic responses while effectively suppressing capacitive background and low-frequency noise. Under optimized conditions, the aptasensor exhibited a linear response over the range of 10-10⁵ PFU mL⁻¹ with a detection limit of approximately 0.25 PFU mL⁻¹ (R² ≈ 0.99). Performance was retained in allantoic fluid, demonstrating robustness in a complex biological matrix. The platform also showed excellent selectivity against non-target influenza subtypes, high reproducibility (RSD < 4%), and stable response over ten days. Collectively, this work establishes a robust, label-free electrochemical strategy for H9N2 detection and provides a modular framework for the sensitive sensing of emerging viral pathogens with strong potential for portable, point-of-care applications.