We report an experimental and computational approach for the fabrication and characterization of a highly sensitive and responsive label-free biosensor that does not require the presence of redox couples in electrolytes for sensitive electrochemical detection. The sensor is based on an aptamer-functionalized transparent electrode composed of nanoporous anodized alumina (NAA) grown on indium tin oxide (ITO)-covered glass. Electrochemical impedance changes in a thrombin binding aptamer (TBA)-functionalized NAA/ITO/glass electrode due to specific binding of α-thrombin are monitored for protein detection. The aptamer-functionalized electrode enables sensitive and specific thrombin protein detection with a detection limit of ∼10 pM and a high signal-to-noise ratio. The transient impedance of the alumina film-covered surface is computed using a computational electrochemical impedance spectroscopy (EIS) approach and compared to experimental observations to identify the dominant mechanisms underlying the sensor response. The computational and experimental results indicate that the sensing response is due to the modified ionic transport under the combined influence of steric hindrance and surface charge modification due to ligand/receptor binding between α-thrombin and the aptamer-covered alumina film. These results suggest that alumina film-covered electrodes utilize both steric and charge modulation for sensing, leading to tremendous improvement in the sensitivity and signal-to-noise ratio. The film configuration is amenable for miniaturization and can be readily incorporated into existing portable sensing systems.
Keywords: anodization; aptasensor; computational electrochemical impedance spectroscopy (EIS); label-free; nanoporous alumina; sensing mechanism.