tetano
Editor, Senior Moderator
Anal Chem
. 2022 Mar 9.
doi: 10.1021/acs.analchem.1c04966. Online ahead of print.
Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples
Isabelle C Samper[SUP] 1 2 [/SUP], Catherine J McMahon[SUP] 1 [/SUP], Melissa S Schenkel[SUP] 1 [/SUP], Kaylee M Clark[SUP] 1 [/SUP], Wisarut Khamcharoen[SUP] 3 [/SUP], Loran B R Anderson[SUP] 4 [/SUP], James S Terry[SUP] 4 [/SUP], Emily N Gallichotte[SUP] 4 [/SUP], Gregory D Ebel[SUP] 4 [/SUP], Brian J Geiss[SUP] 4 5 [/SUP], David S Dandy[SUP] 2 5 [/SUP], Charles S Henry[SUP] 1 2 5 [/SUP]
Affiliations
Abstract
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[SUP]2[/SUP] = 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.
. 2022 Mar 9.
doi: 10.1021/acs.analchem.1c04966. Online ahead of print.
Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples
Isabelle C Samper[SUP] 1 2 [/SUP], Catherine J McMahon[SUP] 1 [/SUP], Melissa S Schenkel[SUP] 1 [/SUP], Kaylee M Clark[SUP] 1 [/SUP], Wisarut Khamcharoen[SUP] 3 [/SUP], Loran B R Anderson[SUP] 4 [/SUP], James S Terry[SUP] 4 [/SUP], Emily N Gallichotte[SUP] 4 [/SUP], Gregory D Ebel[SUP] 4 [/SUP], Brian J Geiss[SUP] 4 5 [/SUP], David S Dandy[SUP] 2 5 [/SUP], Charles S Henry[SUP] 1 2 5 [/SUP]
Affiliations
- PMID: 35263100
- DOI: 10.1021/acs.analchem.1c04966
Abstract
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[SUP]2[/SUP] = 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.