Electrochemical Immunoassay for the Detection of SARS-CoV‑2 Nucleocapsid Protein in Nasopharyngeal Samples

Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2022-03, Vol.94 (11), p.4712-4719
Main Authors: Samper, Isabelle C, McMahon, Catherine J, Schenkel, Melissa S, Clark, Kaylee M, Khamcharoen, Wisarut, Anderson, Loran B. R, Terry, James S, Gallichotte, Emily N, Ebel, Gregory D, Geiss, Brian J, Dandy, David S, Henry, Charles S
Format: Article
Language:English
Subjects:
Analytical chemistry
Antibodies
Chemistry
Coronaviruses
COVID-19
COVID-19 - diagnosis
COVID-19 diagnostic tests
Cross-reactivity
Electrochemistry
Electrodes
Horseradish peroxidase
Humans
Immunoassay
Immunoassay - methods
Infectivity
Influenza
N protein
Nucleocapsid Proteins
Nucleocapsids
Patients
Peroxidase
Polymerase chain reaction
Proteins
SARS-CoV-2
Sensitivity and Specificity
Severe acute respiratory syndrome
Severe acute respiratory syndrome coronavirus 2
Viral diseases
Viruses
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Summary:Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using stencil-printed carbon electrodes (SPCEs) functionalized with capture antibodies targeting the SARS-CoV-2 nucleocapsid protein (N protein). Samples are added to the electrode surface, followed by horseradish peroxidase (HRP)-conjugated detection antibodies also targeting the SARS-CoV-2 N protein. The concentration of the virus in samples is quantified using chronoamperometry in the presence of 3,3′5,5′-tetramethylbenzidine. Limits of detection equivalent to less than 50 plaque forming units/mL (PFU/mL) were determined with virus sample volumes of 20 μL. No cross-reactivity was detected with the influenza virus and other coronavirus N proteins. Patient nasopharyngeal samples were tested as part of a proof-of-concept clinical study where samples were also tested using the gold-standard real-time quantitative polymerase chain reaction (RT-qPCR) method. Preliminary results from a data set of 22 samples demonstrated a clinical specificity of 100% (n = 9 negative samples according to RT-qPCR) and a clinical sensitivity of 70% for samples with RT-PCR cycle threshold (Ct) values under 30 (n = 10) and 100% for samples with Ct values under 25 (n = 5), which complies with the World Health Organization (WHO) criteria for POC COVID-19 diagnostic tests. Our functionalized SPCEs were also validated against standard plaque assays, and very good agreement was found between both methods (R 2 = 0.9993, n = 6), suggesting that our assay could be used to assess patient infectivity. The assay currently takes 70 min from sampling to results.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.1c04966