A COVID19-selective pH-paper test: Ultrafast and highly accurate antibody-free viral detection in native saliva

Abstract

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 has led to high mortality worldwide, with social and financial effects expected to persist for years to come. Since asymptomatic individuals cause a large portion of transmission, actions taken to curb the COVID-19 pandemic mostly rely on procedures aimed at detecting viral-related molecules in infected carriers. Currently, leading available detection methods fall short in reliability or point-of-care mass screening applicability.Here, we present an unprecedented antibody-free smart pH paper-based seconds-long detection platform against the virus-related enzyme 3CLpro as a saliva biomarker of SARS-CoV-2 infection. The selective proteolytic activity of 3CLpro is directly detected from native saliva using a peptide substrate-embedded pH-paper platform. 3CLpro presence in infected individuals leads to an ultrafast pH-drop in the paper matrix, thus leading to a sharp color change as direct means of detection.Ultrafast detection, simplicity, no requirement of surface chemistry by sensitive biomolecules, long-term stability, and point-of-care compatibility for COVID-19 mass screening were demonstrated. Forty-two subjects were tested, exhibiting highly sensitive and specific detection of SARS-CoV-2 infection. Finally, we foresee that this universal approach could be implemented for the multiplexed detection of other viral infections by detecting specific viral proteases.Importantly, since our approach does not require immune-specific antibodies for specific detection, the overall platform is extremely cost-efficient, less than 2 USD cents per assay, and suitable for the worldwide mass screening of viral infections, particularly in remote and developing world locations. Moreover, we propose an extremely easy-to-use and produce practical test strip design.