A-370 Development and clinical study results of a sensitive and specific point-of-care exhaled breath test for respiratory pathogens

Abstract Background A novel electrochemical micro-immunoelectrode (MIE) biosensor utilizing camelid nanobodies has been developed as the sensing element in a point-of-care exhaled breath test for respiratory pathogens, providing results in under 60 seconds from only one deep breath. Methods The MIE comprises a screen-printed electrode specially prepared with a pathogen-specific nanobody, which is covalently bound to the working electrode to concentrate the target pathogen near the surface. Electrical current produced by tyrosine oxidation is measured using square wave voltammetry, and the amount of current produced is proportional to the amount of target bound to the surface. MIE specificity was assessed by comparing peak tyrosine oxidation currents for different concentrations of SARS-CoV-2 and SARS-CoV-1 spike proteins. To determine the biosensor’s limit of detection, a purified inactivated SARS-CoV-2 stock solution was tested in a dilution series at various virus concentrations. To further evaluate system performance, inactivated SARS-CoV-2 virions from three different variants – WA1, Delta, and Omicron (BA.1) – were aerosolized in a controlled laboratory setting in aerosol droplets that mimicked the size distribution of exhaled breath particles. For human studies, the MIE was incorporated into an impaction/condensation surface in a breath collection device. This mechanism accelerates exhaled breath and focuses it onto a chilled surface, where it condenses. This condensate, containing viral particles, is washed onto the biosensor surface for analysis. Results The biosensor is sensitive to 10-40 SARS-CoV-2 viral particles in a sample, depending on the variant. The breath test device was evaluated for sensitivity, specificity, and the number of breaths required to determine illness. A clinical study at the Washington University in St. Louis School of Medicine tested this device for detecting SARS-CoV-2. Thirty-seven patients with respiratory illnesses provided at total of 42 individual breath samples. The results, verified by RT-qPCR for diagnosis, showed 95.5% sensitivity and 94.4% specificity for SARS-CoV-2 over other respiratory pathogens, such as Influenza A, RSV and rhinovirus. Conclusion The breath-based MIE diagnostic platform is a rapid, portable, sensitive, and specific way to identify infected patients using breath. While this study focuses on SARS-CoV-2, the MIE is readily adaptable to other respiratory pathogens, and efforts are currently underway to develop a multiplexed MIE that can detect several different pathogens at once for differential diagnosis of respiratory illness.