Aptamer-Based Activatable Tyramide Signal Amplification for Low-Background Detection of SARS-CoV-2 Nucleocapsid...

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection posed a significant threat to public health and the global economy, in vitro diagnosis of the SARS-CoV-2 nucleocapsid protein proved to be an effective way for SARS-CoV-2 infection control in the past years. Tyramide signal amplification (TSA) has been extensively utilized in tissue imaging and pathological diagnosis owing to the powerful signal enhancement. However, the elevated "ALWAYS ON" fluorescence background limited the accuracy and sensitivity of the conventional TSA assay. To achieve an activated "TURN ON" signal, herein, a small molecule, termed dichlorodihydrofluorescein tyramide (T-DCFH), was synthesized for activatable TSA. Under the catalysis of horseradish peroxidase (HRP) with hydrogen peroxide (H₂O₂), this T-DCFH facilitates the "TURN ON" fluorescence signal. Additionally, as a recognition tool, DNA aptamer has been used for developing in vitro diagnostic approaches. Hence, based on HRP-labeled aptamers binding with SARS-CoV-2 nucleocapsid protein, we achieved aptamers-based activatable TSA detection with a higher signal-to-noise ratio than that of fluorescent dye (FITC)-labeled aptamers, while showing lower background than traditional fluorescein tyramide with "ALWAYS ON". The results demonstrated that the activated T-DCFH significantly enhances the fluorescence signal while diminishing the background noise. By employing multiple aptamers targeting, we offered a timely and accurate in vitro diagnostic approach for future emergent infectious diseases.