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, underscoring the need for rapid and reliable early detection. Here, we report a label-free electrochemical aptasensor based on the B4 anti-H9N2 hemagglutinin (HA) aptamer, implemented for the first time as a biorecognition element in an electrochemical sensing platform. Compared to conventional antibody-based systems, the B4 aptamer offers high binding affinity, improved structural stability, and the ability to recognize intact viral particles, enabling reliable label-free detection. Computational modeling, including molecular docking and molecular dynamics (MD) simulations, was employed to evaluate aptamer–target interactions and to screen candidate sequences against structurally related viral targets, achieving predicted specificity of approximately 90–95%. This approach supports rational aptamer selection and reduces experimental trial-and-error. The sensing interface integrates cerium oxide nanoparticles (CeO₂), electrochemically reduced graphene oxide (rGO), and electrodeposited gold nanoparticles (AuNPs) to form a conductive and stable nanostructured platform. A PolyA–PolyT-modified aptamer enables thiol-free, orientation-controlled immobilization via adenine–gold affinity, improving target accessibility and reducing steric hindrance, while CeO₂ enhances biomolecule adsorption and immobilization stability. Signal transduction was performed using fast Fourier transform square-wave voltammetry (FFT-SWV) within a simplified, miniaturized potentiostat architecture, where part of the signal filtering is transferred from hardware to software. This approach suppresses capacitive background and noise in the frequency domain, improving signal-to-noise ratio and enabling lower detection limits while supporting portable implementation. Under optimized conditions, the aptasensor exhibited a linear response over 1.0 × 10¹ to 1.0 × 10⁵ PFU mL⁻¹ with a detection limit of 0.25 PFU mL⁻¹ (R² ≈ 0.99). The sensor demonstrated high selectivity, excellent reproducibility (RSD < 4%), and stable performance over ten days, with retained performance in allantoic fluid, confirming robustness in complex biological matrices. This work presents a sensitive and modular electrochemical sensing platform that integrates computationally guided aptamer design, engineered nanointerfaces, and advanced signal processing, offering strong potential for portable and point-of-care viral detection.