Demonstration and structural basis of a therapeutic DNA aptamer for SARS-CoV-2 spike protein detection

At the onset of the COVID-19 pandemic, the absence of rapid and precise diagnostic tools hindered early detection and response. To address this challenge, we developed a renewable electrochemical impedance biosensor (aptasensor) using a therapeutic DNA aptamer immobilized on a nanostructured gold nanoparticle/carbon nanotube (AuNP/CNT) electrode to detect the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). The aptasensor achieved a limit of detection of 0.19 pg mL^-1 and a dynamic range from 1 to 10⁵ pg mL^-1. Following regeneration with a 60-s pH 2.0 rinse, the sensor retained over 90% of its original signal across five cycles and remained stable after two weeks of ambient storage. Dual-mode readouts, utilizing impedance spectroscopy and surface plasmon resonance (SPR), confirmed binding specificity and reproducibility. Cryogenic electron microscopy (cryoEM) resolved the aptamer-S protein complex in the open conformation, revealing a bridge-like interaction with conserved residues Y489, N487, F486, and S477. These contacts remained functional despite Omicron BA.2 mutations (S477N, N501Y) and aligned with previously reported mutational data. Specificity was further supported by negative controls and structural consistency with known hACE2 binding footprints. These results establish a robust, low-cost biosensor platform combining reuse, structural insight, and variant tolerance. The aptasensor's scalability and adaptability make it a strong candidate for future diagnostic applications targeting evolving viral threats.