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

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 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.

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

“…Long-term future work should determine the LOD by serial dilution measurements to have a more realistic understanding of the sensors’ capabilities at low concentrations. While the drying process as opposed to incubation may have resulted in a slight loss of signal compared to the method our group reported before, 62 this method is much more practical heading toward manufacturing and its working range and LOD are still sufficient for most COVID-19 cases, which have an extremely wide range of concentration from 5 to 10 6 equivalent PFU/mL. 67 Higher infectivity has also been shown to be correlated with higher equivalent PFU/mL values.…”

“…Our group previously reported a method in which the capture antibody and aged casein were incubated for 1 h each and required manual washing steps between incubations. 62 Initially, the same incubation and washing steps were used to modify the electrode before incorporation into the device. While there were significant signal differences between the blank and SARS-CoV-2 (representative amperograms shown in Figure SI3 ), these steps were not practical for the long-term goals of the device.…”

Investigating transmission of SARS-CoV-2 using novel face mask sampling: a protocol for an observational prospective study of index cases and their contacts in a congregate setting

“…3 One of our aims is to validate a novel antigen assay for testing PVA strips from the masks. For this assay, electrodes were prepared according to Samper et al 21 For sample analysis, 20 µL of PVA mask sample or virus standard was added to the functionalised electrode and incubated for 40 min, and then rinsed with PBST (phosphate buffer saline with tween) and PBS(phosphate buffer saline) using solid stream spray bottles. Virus standards were inactivated with 0.1% Igepal and diluted in Hanks' Balanced Buffer Solution with 0.1% Tween80 and 0.1% Igepal.…”

Progress in Electrochemical Biosensing of SARS-CoV-2 Virus for COVID-19 Management

“…Therefore, attempts were made to develop highly sensitive electrochemical biosensing systems based on an antibody, aptamer, and MIP receptors for detecting SARS-CoV-2 NP. Samper et al developed a highly sensitive and label-based electrochemical immunoassay for quantitatively detection of SARS-CoV-2 NP using stencil-printed carbon electrodes (SPCEs) (Figure 6a) [89]. The abundant carboxyl groups (-COOH) on the SPCEs enabled the immobilization of anti-SARS-CoV-2 NP via EDC/NHS coupling.…”