Detection of Viruses through a Steric Hindrance Aptamer Assay Coupled with Electrochemical Readout

The urgent need for rapid infectious disease tests that effectively combine sensitivity, specificity, speed, and ease of operation is increasingly recognized. Aptamers present a promising solution for the specific identification of infectious disease targets, such as viruses; however, the development of aptamer-based assays poses significant challenges. In the realm of electrochemical readout, these challenges stem from the difficulty of translating target capture into an electrochemical signal, particularly when large viral targets hinder the application of structure-switching aptamers or sandwich assays. In this study, we introduce the steric hindrance aptamer assay, which leverages viral capture to enhance steric hindrance between two complementary DNA strands, thereby modulating the electrochemical signal. This innovative assay achieves a limit of detection of 89 copies/mL in buffer and 1505 copies/mL in saliva for the identification of SARS-CoV-2 Omicron pseudovirus and 364 and 1704 copies/mL for detecting influenza A in buffer and saliva, respectively. Furthermore, this assay is evaluated using 20 clinical samples (10 positives and 10 negatives), successfully identifying patients infected with SARS-CoV-2 with 100% sensitivity and specificity when compared to reverse transcription quantitative polymerase chain reaction. The reported viral analysis is achieved within a timeframe of less than 1 h while maintaining high specificity against other respiratory viruses.