In a recent article published in the journal ACS Sensors, a group of researchers developed a rapid, non-invasive, point-of-care platform for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exhaled breath, potentially adaptable for multiple respiratory virus detection.
Infectious respiratory viruses like SARS-CoV-2, influenza, rhinovirus, and respiratory syncytial virus (RSV) predominantly spread via inhaled aerosols emitted by infected individuals.
These aerosols contain a variety of sizes of viral ribonucleic acid (RNA), with the highest viral loads detected in aerosols smaller than 1 μm. Despite the importance of aerosol transmission, real-time detection techniques remain limited.
Currently, screening for SARS-CoV-2 RNA in breath aerosols involves a lengthy process that requires sophisticated equipment and trained personnel. While electrochemical biosensors have been used for virus detection, their use has been primarily for nasal swabs, saliva, or sputum samples. With the emergence of new SARS-CoV-2 variants, there is a pressing need for rapid, variant-sensitive testing methods.
About the study
In the present study, the testing platform design incorporates a breath aerosol collection mechanism and a micro-immunoelectrode (MIE) biosensor.
The device collects breath aerosols using a 3D-printed polylactic acid box equipped with a cap featuring an inlet straw and two injection ports for liquids. This box houses a polyimide film coated with silicon wax, providing an inclined hydrophobic surface to facilitate aerosol collection.
When an individual exhales into the device, aerosols, potentially bearing viral particles, condense on the cooled surface. Subsequent washing with bovine serum albumin in phosphate buffer saline enables the collection of the condensed aerosols, which were then analyzed by the MIE biosensor.
The MIE biosensor uses cost-effective screen-printed carbon-based electrodes pretreated to enhance tyrosine oxidation and attachment of a SARS-CoV-2 specific nanobody. This procedure guarantees that any existing tyrosine in the nanobody gets oxidized during electrode preparation and cannot create a signal during the actual test.
In the initial prototype stage, the breath samples were diluted in a separate solution before analysis. When fully functional, the biosensor connects to a potentiostat, and square-wave voltammetry was performed to detect SARS-CoV-2.
The authors planned to create a reusable version of the breath aerosol analyzer with an integrated decontamination and cleaning mechanism.
The electrodes were pretreated to enhance tyrosine oxidation and efficient nanobody binding. After the nanobody was attached, the biosensors were treated with ethanolamine and albumin to deactivate reactive amine sites and block non-specific protein binding sites.
The authors also performed laboratory experiments, including cell and virus culture, aerosolization, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and clinical studies, to validate their platform and methods.
Through these experiments, they simulated exhalation conditions, generated aerosols that mimic breath from the lower airways, measured viral load in the breath samples, and evaluated their diagnostic setup in human subjects.
In the present study, the MIE biosensor's specificity was examined by contrasting the peak tyrosine oxidation currents (Iox) for varied concentrations of SARS-CoV-1 and SARS-CoV-2 spike proteins.
The latter rendered a strong signal as low as 20 pg of spike protein/mL of the sample fluid and plateaued around 20 ng/mL. In contrast, SARS-CoV-1 resulted in a minimal signal, underscoring the biosensor’s high specificity towards SARS-CoV-2, despite the substantial genetic similarities between the two spike proteins.
Further, the MIE biosensor’s limit of detection (LoD) was evaluated by a sequential dilution of an inactivated SARS-CoV-2 stock solution, followed by the measurement of Iox values for diverse virus concentrations.
The lowest detected virus RNA concentrations were 32, 8, 6, and 21 RNA copies per mL for the USA/WAa1/2020, β, Delta, and Omicron strains of SARS-CoV-2, respectively. Notably, the LoD for all the variants was significantly lower than the usual viral RNA load found in exhaled breaths of individuals with SARS-CoV-2. This affirms the biosensor's potential in detecting virus aerosols in exhaled breath with ultra-sensitivity.
To appraise the device's efficiency, laboratory experiments aerosolized inactive SARS-CoV-2 virions of three distinct variants: WA1, Delta, and Omicron. Aerosols mimicking the size distribution of exhaled breath from the lungs' lower airways were generated.
Control experiments used pure phosphate-buffered saline (PBS) solution. A 77.8% sensitivity was demonstrated for the device, comparing favorably with other electrochemical detection techniques.
The viral RNA amounts from the breath aerosol analyzer samples were within the range seen in other studies using exhaled breath condensate (EBC)-based methods. The viral loads were significantly lower than those obtained from nasal swabs or saliva samples.
Despite this, the potential for virus detection in exhaled breath was confirmed, given the estimated 200–600 viral particles per breath in a coronavirus disease 2019 (COVID-19) infection.
To substantiate the system's efficacy in humans, a clinical trial was conducted at the Infectious Disease Clinical Research Unit, Washington University School of Medicine, involving eight participants.
Considerations were given to parameters such as typical viral loads in infected patients, device collection efficacy, and LoD of the MIE biosensor when determining the required breaths for adequate sample collection.
Each participant blew into the device 2, 4, and 8 times with about 3 minutes between each sample collection. The biosensor revealed a 77.9% sensitivity and a 100% specificity as the analyte signal for both negative patients fell below the LoD.
The in vitro biosensor test utilized known inactivated viral particles through the BA.1 variant, while the clinical study was conducted when the BQ.1 variant was predominant. Nonetheless, the sequencing of the variant in the human subjects was not carried out.
To summarize, the authors developed an affordable, portable testing platform that featured a breath aerosol collector and nanobody-based MIE biosensor. With results in under a minute, this non-invasive, easy-to-use method required minimal training.
From just 20 seconds of breath, it could effectively detect SARS-CoV-2. The biosensor's sensitivity surpassed similar devices, and it could adapt to detect different virus variants or other pathogens.