Conventional electrochemical biosensing systems rely on a single output signal, which limits their certain practical application, specifically from the viewpoint of external interference factors causing electrochemical signal errors. This study reports a self-calibrating dual-electrode based electrochemical aptasensor for the reliable and independent detection of avian influenza viruses (AIVs), which are the primary cause of highly contagious respiratory diseases, under external interference factors. Both electrodes were fabricated using tungsten rods surface-modified with a 3D nanostructured porous silica film (3DNRE). Subsequently, methylene blue (MB) was loaded as a redox-active material into the pores and capped with corresponding aptamer. One electrode was capped with an anti-AIV nucleoprotein (NP) aptamer (AptAIV-MB@3DNRE) allowing target-specific binding, resulting in changes in electrochemical signal upon diffusional release of the loaded redox molecules. The other electrode was capped with a control aptamer (Aptcon-MB@3DNRE), serving as a reference to correct false responses generated by nonspecific aptamer detachment and MB release under environmental changes in pH and ion strength and presence of nontarget molecules from cell lysis debris. In the dual-electrode platform, Aptcon-MB@3DNRE provides a corrected baseline for the fluctuating original output signals from AptAIV-MB@3DNRE. Consequently, this dual-electrode platform exhibits excellent output-signal stability (relative standard deviation, RSD: 5.86%) compared to a conventional single-electrode platform (RSD: 30.13%) at equivalent concentrations of AIV NP samples under different reaction buffer conditions. Moreover, no further purification and washing steps were required, indicating that the strategy may represent a universal and reliable platform for the electrochemical aptamer-based detection of various biomolecules.

中文翻译：

---

自校准电化学适体平台：纠正外部干扰错误，以可靠，稳定地检测禽流感病毒。

常规的电化学生物传感系统依赖于单个输出信号，这限制了它们的某些实际应用，特别是从引起电化学信号错误的外部干扰因素的角度来看。这项研究报告了一种基于自校准双电极的电化学适体传感器，可在外部干扰因素下可靠且独立地检测禽流感病毒（AIV），这是高度传染性呼吸道疾病的主要原因。两个电极均使用钨棒制成，该钨棒经3D纳米结构多孔二氧化硅膜（3DNRE）表面改性。随后，将亚甲基蓝（MB）作为氧化还原活性材料加载到孔中，并用相应的适体封端。一个电极上盖有抗AIV核蛋白（NP）适体（AptAIV-MB @ 3DNRE），可实现靶标特异性结合，导致负载的氧化还原分子扩散释放后电化学信号发生变化。另一个电极用对照适体（Aptcon-MB @ 3DNRE）盖住，作为校正因pH和离子强度的环境变化以及细胞裂解碎片中存在非目标分子而引起的非特异性适体脱离和MB释放所产生的错误反应的参考。在双电极平台中，Aptcon-MB @ 3DNRE为来自AptAIV-MB @ 3DNRE的波动原始输出信号提供了校正后的基线。因此，与传统的单电极平台（RSD：30）相比，该双电极平台具有出色的输出信号稳定性（相对标准偏差，RSD：5.86％）。在不同反应缓冲液条件下以等浓度的AIV NP样品（13％）洗脱。而且，不需要进一步的纯化和洗涤步骤，表明该策略可以代表用于各种生物分子的基于电化学适体的检测的通用且可靠的平台。