(443d) Point-of-Care Detection of Respiratory Virus Aerosols in Exhaled Breath

Airborne transmission via virus-laden aerosols is a dominant route for the transmission of respiratory diseases, including SARS-CoV-2. Developing techniques for real-time detection of respiratory virus aerosols remains a longstanding technical challenge. The current state of research involves exhaled breath condensate (EBC) sample collection followed by reverse transcription-polymerase chain reaction (RT-PCR) to detect the prevalence of SARS-CoV-2. This method has limitations in mass testing applications due to lengthy turnaround times and the need for trained personnel. Recently, electrochemical biosensors are emerging as an alternative to standard clinical screening techniques as they are rapid and possess a low detection limit. However, very few studies have demonstrated the use of electrochemical biosensors to detect SARS-CoV-2 in EBC samples.

Here, we introduce a non-invasive, point-of-care testing platform that directly detects SARS-CoV-2 aerosols in as little as two exhaled breaths of patients and will generate test results in under 60 seconds. The platform integrates a hand-held breath aerosol collection device and a llama-derived nanobody specific to SARS-CoV-2 spike-protein bound to an ultrasensitive micro-immunoelectrode (MIE) biosensor. The breath aerosol collector has an inlet straw through which a patient exhales into the device. Virus-laden respiratory aerosols from the exhaled breath impact, condense on the chilled hydrophobic surface and slide along the tapered incline to the bottom corner of the box, where the MIE biosensor is located. The MIE biosensor detects the oxidation of tyrosine amino acids present in the spike protein of SARS-CoV-2. It is connected to a commercial potentiostat and square wave voltammetry is performed to oxidize tyrosine and measure the peak oxidation current corresponding to the presence of virus aerosols in a given sample.

We evaluated our device performance by aerosolizing inactivated SARS-CoV-2 virions of three different variants: WA1, Delta, and Omicron (BA.1) in laboratory experiments. We generated aerosols that mimic the size distribution of exhaled breath originating from the lower airways of lungs. Additionally, to validate the performance of our system in human patients, we employed our device in a clinical trial study and analyzed EBC samples of 8 patients. Lab experiments and clinical trial data indicate a device sensitivity of about 80%, and the MIE biosensor is highly specific to SARS-CoV-2. The results from RT-qPCR determined that viral RNA for samples collected using our device ranged from 10^1.3 to 10^3.7 gene copies/sample. The device is successful in detecting all variants of concern up to omicron BA.5. The MIE biosensor is sensitive to ~10 viral particles in a sample (as determined by RT-qPCR) and directly detects the virus itself as opposed to a signature/pattern of the virus. We obtained adequate results from a sampling time of just 20-30 seconds (2 exhaled breaths) as compared to 5-30 minutes in typical EBC-based studies. Our platform in the future could also be multiplexed for detection of other respiratory pathogens of interest. It provides a rapid and non-invasive alternative to conventional viral diagnostics, and is highly promising for mass testing applications in places like airports and conference centres. Current work is focused on in-house fabrication of screen-printed carbon electrodes (SPCEs) using an affordable manual screen-printing method. A mixture of carbon conductive ink and graphene is used for the working and counter electrodes, and Ag/AgCl ink for the reference electrode. Electrochemical sensors have limitations pertaining to low shelf life, and we intend to have in-house ability for developing low-cost sensing systems and testing.