Real-time environmental surveillance of SARS-CoV-2 aerosols

The COVID-19 pandemic, caused by SARS-CoV-2, continues to pose a global challenge. Airborne transmission is a significant infection pathway, leading to rapid spread. Governments implemented various control measures (masking, quarantining, social distancing), but these significantly impacted daily life. The lack of rapid, affordable community-level infection detection protocols hindered effective disease control. A real-time, non-invasive surveillance device for SARS-CoV-2 aerosols is crucial for improved infection management. Offline air sampling techniques (using samplers like condensation growth-based particle into liquid samplers (PILS), wet-wall cyclone-based PILS, and filter sampling, followed by RT-qPCR) have been used for SARS-CoV-2 detection in various environments. However, these methods have limitations: long turnaround times (1–24 h), requirement of skilled labor, and lack of real-time information. The absence of commercially available automated real-time airborne SARS-CoV-2 detection devices stems from two technological gaps: the need for an efficient high-flow virus aerosol sampler integrable into a real-time detector, and the need for a fast, accurate, and sensitive virus detection protocol capable of measuring low virus concentrations in ambient air. High flow rate samplers improve virus recovery, and biosensors offer a promising alternative to RT-qPCR due to their speed, sensitivity, and cost-effectiveness. However, no previous studies had integrated high-flow sampling with biosensors for real-time airborne SARS-CoV-2 detection.

Existing literature highlights the challenges in real-time SARS-CoV-2 aerosol detection. Studies using offline methods like RT-qPCR on samples collected by various air samplers demonstrated the presence of SARS-CoV-2 RNA in diverse settings. However, these methods lack real-time capabilities. Research on high-flow air samplers showed improved virus recovery at higher flow rates, but integration with real-time detection remained a challenge. Biosensors have emerged as a potential solution for rapid and sensitive pathogen detection, but their application to airborne SARS-CoV-2 was largely unexplored before this study. The reviewed literature underscored the need for a technology that combines efficient aerosol sampling with rapid, sensitive, and specific detection for real-time monitoring.

This study introduces a pathogen Air Quality (pAQ) monitor that combines a custom high-flow batch-type wet-wall cyclone PILS with a llama-derived nanobody-based micro-immunoelectrode (MIE) biosensor. The wet cyclone operates at ~1000 lpm, collecting aerosols into a liquid medium (PBS). This solution is then transferred to the MIE detection unit. The MIE biosensor, adapted from a technology used for amyloid-β detection, utilizes screen-printed carbon electrodes with covalently attached nanobodies that specifically bind to the SARS-CoV-2 spike protein. Square wave voltammetry (SWV) is performed to detect the oxidation of tyrosine amino acids in the spike protein, providing a signal proportional to the virus concentration. The wet cyclone's performance was compared to commercially available low-flow PILS (BioSampler and Liquid Spot Sampler) through chamber experiments with aerosolized inactivated SARS-CoV-2 (WA-1 strain). The pAQ monitor's sensitivity and limit of detection (LoD) were evaluated using various inactivated SARS-CoV-2 variants (WA-1, delta, beta, and BA-1). Additionally, real-world testing was conducted by deploying the pAQ monitor in the apartments of two SARS-CoV-2-positive patients.

The wet cyclone demonstrated comparable or superior virus sampling performance to commercial samplers. In chamber experiments, the wet cyclone showed significantly higher viral RNA recovery, particularly at low concentrations. The pAQ monitor achieved a sensitivity of 77% for WA-1 and 83.3% for BA-1. The LoD varied among SARS-CoV-2 variants (35, 7, 9, and 23 RNA copies/m³ for WA-1, delta, beta, and BA-1, respectively), likely due to variant-specific mutations affecting nanobody binding. Real-world testing in the apartments of SARS-CoV-2-positive individuals yielded positive RT-qPCR results for all air samples collected using the wet cyclone, indicating the presence of SARS-CoV-2 RNA, albeit at low concentrations (high Ct values). The findings show that the wet cyclone is efficient in collecting virus particles even in low concentration scenarios, making it suitable for use in real-world environments. The high sensitivity, low limit of detection, and real-time monitoring capabilities of the pAQ monitor make it suitable for real-time detection of SARS-CoV-2 in various indoor environments.

The results demonstrate the feasibility of real-time airborne SARS-CoV-2 detection using the pAQ monitor. The high virus capture efficiency of the wet cyclone, combined with the sensitivity and speed of the MIE biosensor, addresses the technological gaps identified in existing methods. The ability to detect SARS-CoV-2 in real-time, even at environmentally relevant low concentrations, provides a valuable tool for rapid disease control. The success in detecting SARS-CoV-2 RNA in the homes of infected individuals further validates its potential for real-world deployment. The observed variation in LoD across SARS-CoV-2 variants highlights the need for further optimization and potentially variant-specific nanobodies. However, the overall performance suggests the pAQ monitor is a significant advancement in environmental surveillance technologies.

This study successfully demonstrated a proof-of-concept pAQ monitor for real-time SARS-CoV-2 aerosol detection. The integration of a high-flow wet cyclone with an ultrasensitive MIE biosensor provided high sensitivity and a low limit of detection. The successful field testing in infected households validated the system's potential for real-world applications. Future work will focus on noise reduction, multiplexed detection of other pathogens, and comprehensive testing in diverse environments to assess robustness and identify potential interfering agents.

The current pAQ monitor design has a high noise level (75-80 dB), requiring further development for noise reduction to improve user experience. The LoD varied slightly between different SARS-CoV-2 variants, suggesting potential improvements in nanobody design or assay optimization could be considered. The study's real-world testing was limited to two households, requiring more extensive field testing to confirm the technology's robustness and generalizability across diverse environments with varying aerosol compositions. Further investigation is needed to determine the potential impact of various interfering agents in different air samples on the biosensor performance.