The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 μL sample with a pipette. The device, which we call the μSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2–3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus ( n = 62). Moreover, the dynamic range of the sensor extends at least from 10 5.9 virions per mL to 10 10.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (10 5.6 –10 7 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.
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The size and culturability of patient-generated SARS-CoV-2 aerosol
Abstract Background Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. Objective We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. Methods Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). Results SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. Significance Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.
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- Award ID(s):
- 2032135
- PAR ID:
- 10296888
- Date Published:
- Journal Name:
- Journal of Exposure Science & Environmental Epidemiology
- ISSN:
- 1559-0631
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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