A Real-Time Enzyme-Linked Aptamer Assay (RT-eELAA) for Rapid and Sensitive Detection of Viral Antigens in Saliva Using Electrochemical Readout

Despite the need for reliable rapid antigen tests for infectious disease diagnostics, tests that combine rapid answer-to-result times with high sensitivity and specificity remain elusive. A major challenge in developing such tests is the loss of performance of analytical assays in clinical samples. Herein, we developed a rapid antigen test based on a real-time electrochemical sandwich assay for detecting SARS-CoV-2 and Influenza A in saliva. This assay used aptamers for both target capture and signal transduction and produced limits of detection of 301 and 743 copies/mL for SARS-CoV-2 and Influenza A, respectively. When evaluating this assay with clinical saliva samples, we encountered major issues in distinguishing between positive and negative samples. In response, we developed a revised method to interrogate each clinical sample with a pair of electrochemical detectors modified, respectively, with a functional aptamer or a nonfunctional aptamer mutant. This method enabled us to normalize the signal response measured from each clinical sample with a reference signal, overcoming the previously encountered challenge and resulting in a clinical sensitivity of 100% and a specificity of 100% when analyzing 20 saliva samples that were collected and tested for COVID-19.