An interface-engineered signal-on electrochemical aptasensor for H5N2 avian influenza virus detection

Outbreaks of avian influenza virus (AIV) cause substantial economic losses in the poultry industry, highlighting the need for sensitive, reliable and field-adaptable detection platforms. In this study, we report a signal-on electrochemical aptasensor for avian influenza virus H5N2(K08-404) by translating a previously reported J3/JH4 cognate aptamer pair into an electrode-confined sandwich sensing format. The simultaneous binding capability of this aptamer pair enabled target-mediated recruitment of the HRP-labeled reporter aptamer and generated a signal-on amperometric response. This signal-on aptasensor was successfully fabricated by immobilizing the primary aptamer on the screen-printed gold electrode followed by the addition of a mixture of the target virus and the secondary aptamer labeled with horseradish peroxidase, thereby generating amplified signal-on electrochemical responses. The fabrication of the sandwich-type aptasensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Decreased peak currents and increased peak-to-peak separation supported the stepwise assembly of the electrochemical sensing interface. Analytical performance was evaluated at various virus concentrations using chronoamperometry, revealing a concentration-dependent increase in current response. The aptasensor achieved LODs of 2.00 × 10⁴ EID₅₀ per mL in a buffer and 2.76 × 10⁴ EID₅₀ per mL in a processed duck fecal matrix, with linear working ranges of 3.91 × 10⁴-4.17 × 10⁵ EID₅₀ per mL and 1.95 × 10⁴-1.25 × 10⁶ EID₅₀ per mL, respectively. These results support the feasibility of converting a previously validated H5N2 aptamer pair into a quantitative electrochemical platform for surveillance-oriented virus detection.