Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 30;8(39):eabn9665.
doi: 10.1126/sciadv.abn9665. Epub 2022 Sep 28.

Microfluidic affinity selection of active SARS-CoV-2 virus particles

Sachindra S T Gamage  1   2 , Thilanga N Pahattuge  1   2 , Harshani Wijerathne  1   2 , Katie Childers  2   3 , Swarnagowri Vaidyanathan  2   3 , Uditha S Athapattu  1   2 , Lulu Zhang  2   3 , Zheng Zhao  1   2 , Mateusz L Hupert  4 , Rolf M Muller  4 , Judy Muller-Cohn  4 , Janet Dickerson  4 , Dylan Dufek  4 , Brian V Geisbrecht  5 , Harsh Pathak  6 , Ziyan Pessetto  7 , Gregory N Gan  8   9 , Junseo Choi  2   10 , Sunggook Park  2   10 , Andrew K Godwin  2   6   9 , Malgorzata A Witek  1   2 , Steven A Soper  1   2   3   9   11
Affiliations

Microfluidic affinity selection of active SARS-CoV-2 virus particles

Sachindra S T Gamage et al. Sci Adv. .

Abstract

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus's S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip's surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Viral load profile and variant frequency in Kansas and Missouri.
(A) Hypothetical viral load as a function of disease progression and different testing strategies. From (2) New England Journal of Medicine, M. J. Mina, R. Parker, D. B. Larremore, Rethinking Covid-19 test sensitivity—a strategy for containment, vol. 383, pg. e120. Copyright © (2020) Massachusetts Medical Society. Reprinted with permission from the Massachusetts Medical Society. (B) Frequency of appearance of different SARS-CoV-2 variants identified in Kansas and northeast Missouri between November 2020 and May 2022. Variants are presented as clade and normalized to 100% at each time point. Data are found at www.gisaid.org/phylodynamics/global/nextstrain/. (C) Schematic showing the workflow of the reported assay. Saliva samples [COVID-19(+) or COVID-19(−)] are flowed through a microfluidic chip containing an aptamer (affinity agent) that is attached to the surface of the chip through a heterobifunctional linker that can be cleaved using blue light. Following release of intact VPs, the selected particles can be characterized via atomic force microscopy (AFM), transmission electron microscopy (TEM), or nanoparticle tracking analysis (NTA) and subjected to RT-qPCR. In this case, the microfluidic chip selects active VPs that have an accessible ACE2 research binding domain in the S protein.
Fig. 2.
Fig. 2.. The VP selection chip and covalent attachment of the affinity agent.
(A) Micrographs of the VP selection chip, cover plate, and assembled VP selection chip. (B) Scanning electron microscopies (SEMs) of several selection beds in the VP selection chip. Shown is the fluidic input/output feed network into several beds and a high-resolution SEM of one bed with its micropillars. (C) Summary of the operational characteristics of the VP selection chip. (D) Rapid scanning confocal image of a section of the VP selection chip. (E) Topographical profile of the micropillars and interpillar spacing in the VP selection chip shown in (D). (F) Production line of the injection molded VP selection chips. (G) Scheme demonstrating covalent attachment of the aptamer via the PC linker to the UV/O3-activated COP surface of the plastic chip. Also shown is the secondary structure of the 51–nucleotide (nt) SARS-CoV-2 aptamer (see electrospray ionization for detailed description of this secondary structure).
Fig. 3.
Fig. 3.. SPR of SARS-CoV-2 binding to affinity agent.
(A) Sensogram showing the binding kinetics of recombinant SARS-CoV-2 S protein (RBD, rabbit Fc Tag, yellow object) to the SARS-CoV-2 aptamer. (B) Concentration isotherm for binding of the RBD S protein to its 51-nt aptamer. The control consisted of a channel with no aptamer. (C) Sensograms of SARS-CoV-2 VP binding kinetics to a specific (51-nt SARS-CoV-2) and nonspecific [human respiratory syncytial virus (HRSV)] aptamer. (D) Concentration isotherm of binding of heat-inactivated SARS-CoV-2 VP to its 51-nt aptamer. The control consisted of a channel with a random DNA sequence aptamer. The negative RU (blue line) is indicative of a negative bulk refractive index shift and lack of binding to the surface. The VP concentration varied between 3.6 × 106 and 45 × 106 genome equivalents of RNA per milliliter.
Fig. 4.
Fig. 4.. VP selection chip characterization and selection specificity.
(A) Box plots representing SARS-CoV-2 and HRSV nonspecific binding to HRSV and SARS-CoV-2 aptamers, respectively, and recovery of VPs from buffer and saliva to their specific aptamers bound to the affinity bed at different linear flow velocities used for sample processing. (B) Summary of the recovery of different VPs to the VP selection chip using different aptamers. (C) AFM image of a selected and subsequently released SARS-CoV-2 particles using the VP selection chip. (D) NTA analysis of a stock solution of heat-inactivated SARS-CoV-2 (red trace) and selection and photo-released SARS-CoV-2 VPs from the chip (black trace). (E) UV-vis transmission spectra for COC (cyclic olefin copolymer) and COP plates (2 mm in thickness). LED output light range is shown as a reference. (F) Absorbance of a 1% PVP/0.5% BSA solution and PBS buffer in the UV-vis range. Absorbance spectrum measured in a 1-cm path length cuvette. au, arbitrary units.
Fig. 5.
Fig. 5.. VP selection assay for the analysis of clinical samples.
(A) Summary of results for saliva samples secured from anonymous donors. (1) Healthy donors test were performed by Sinochips Diagnostics with 0.2 ml of saliva sample. COVID-19–positive individuals’ status was confirmed using the Cepheid Xpress SARS-CoV-2 test from nasopharyngeal swabs. (2) A saliva sample of 0.2 ml was processed using the 51-nt SARS-CoV-2 aptamer–modified SARS-CoV-2 VP selection chip. Ten microliters of the photo-released VPs were evaluated by RT-qPCR. N1 and/or N2 are virus nucleocapsid (N) gene fragments targeted for specific detection of SARS-CoV-2 VPs. (3) A Roche-based antibody test was performed at Sinochips Diagnostics, Olathe, KS. (4) Approximately 0.2 ml of the saliva flow through from the microfluidic chip was evaluated by RT-qPCR. N1 and/or N2 are virus nucleocapsid (N) gene fragments targeted for specific detection of SARS-CoV-2. (5) Approximately 20 μl of the effluent was heat-inactivated. (B) Experimental design for evaluating the 30 clinical samples received for this study. Matched nasopharyngeal and saliva samples were secured for each of the 30 patients through an IRB-approved protocol at the University of Kansas Medical Center. (C) SEM of the five–in-plane nanopore focused ion beam milled into a Si wafer. (D) SEM showing a bridge channel flanking the access microchannels. The in-plane nanopore is positioned at the input side of the bridge channel shown in the figure. The SEM shown here is the plastic chip made in COP that was fabricated by imprinting. (E) High-resolution SEM showing an in-plane nanopore imprinted into a COP plastic chip, which was designed to have an approximate 350-nm effective diameter. (F) nCC transient current traces for heat-inactivated SARS-CoV-2 VPs.
See this image and copyright information in PMC

References

    1. Ke Z., Oton J., Qu K., Cortese M., Zila V., McKeane L., Nakane T., Zivanov J., Neufeldt C. J., Cerikan B., Lu J. M., Peukes J., Xiong X., Kräusslich H.-G., Scheres S. H. W., Bartenschlager R., Briggs J. A. G., Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature 588, 498–502 (2020). - PMC - PubMed
    1. Mina M. J., Parker R., Larremore D. B., Perspective: Rethinking Covid-19 test sensitivity—A strategy for containment. N. Engl. J. Med. 383, e120 (2020). - PubMed
    1. CDC, COVID-19. (2021), vol. 2021; https://www.cdc.gov/coronavirus/2019-ncov/index.html.
    1. Hogan C. A., Garamani N., Lee A. S., Tung J. K., Sahoo M. K., Huang C., Stevens B., Zehnder J., Pinsky B. A., Comparison of the Accula SARS-CoV-2 test with a laboratory-developed assay for detection of SARS-CoV-2 RNA in clinical nasopharyngeal specimens. J. Clin. Microbiol. 58, e01072-20 (2020). - PMC - PubMed
    1. Creager H. M., Cabrera B., Schnaubelt A., Cox J. L., Cushman-Vokoun A. M., Shakir S. M., Tardif K. D., Huang M.-L., Jerome K. R., Greninger A. L., Drobysheva D., Spaulding U., Rogatcheva M., Bourzac K. M., Hinrichs S. H., Broadhurst M. J., Fey P. D., Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2. J. Clin. Virol. 129, 104538 (2020). - PMC - PubMed