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 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 (R2 = 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.

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics.

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15-20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Electrochemical Capillary Driven Immunoassay for Detection of SARS-CoV-2.

The COVID-19 pandemic focused attention on a pressing need for fast, accurate, and low-cost diagnostic tests. This work presents an electrochemical capillary driven immunoassay (eCaDI) developed to detect SARS-CoV-2 nucleocapsid (N) protein. The low-cost flow device is made of polyethylene terephthalate (PET) and adhesive films. Upon addition of a sample, reagents and washes are sequentially delivered to an integrated screen-printed carbon electrode for detection, thus automating a full sandwich immunoassay with a single end-user step. The modified electrodes are sensitive and selective for SARS-CoV-2 N protein and stable for over 7 weeks. The eCaDI was tested with influenza A and Sindbis virus and proved to be selective. The eCaDI was also successfully applied to detect nine different SARS-CoV-2 variants, including Omicron.