Microfluidic Magneto Immunosensor for Rapid, High Sensitivity Measurements of SARS-CoV-2 Nucleocapsid Protein in Serum

Abstract

The COVID-19 pandemic has highlighted the importance and urgent need for rapid and accurate diagnostic tests for COVID-19 detection and screening. The objective of this work was to develop a simple immunosensor for rapid and high sensitivity measurements of SARS-CoV-2 nucleocapsid protein in serum. This assay is based on a unique sensing scheme utilizing duallylabeled magnetic nanobeads for immunomagnetic enrichment and signal amplification. This immunosensor is integrated onto a microfluidic chip, which offers the advantages of minimal sample and reagent consumption, simplified sample handling, and enhanced detection sensitivity. The functionality of this immunosensor was validated by using it to detect SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 50 pg/mL in whole serum and 10 pg/mL in 5× diluted serum. We also adapted this assay onto a handheld smartphone-based diagnostic device that could detect SARS-CoV-2 nucleocapsid protein at concentrations as low as 230 pg/mL in whole serum and 100 pg/mL in 5× diluted serum. Lastly, we assessed the capability of this immunosensor to diagnose COVID-19 infection by testing clinical serum specimens, which revealed its ability to accurately distinguish PCR-positive COVID-19 patients from healthy, uninfected individuals based on SARS-CoV-2 nucleocapsid protein serum levels. To the best of our knowledge, this work is the first demonstration of rapid (<1 h) SARS-CoV-2 antigen quantification in whole serum samples. The ability to rapidly detect SARS-CoV-2 protein biomarkers with high sensitivity in very small (<50 μL) serum samples makes this platform a promising tool for point-of-care COVID-19 testing.

Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen

“…Th manufacturer of the antibody used in this study (Sino Biological) has also reported cross reactivity with SARS-CoV. This cross reactivity has been previously reported for all the immunoassays using the N protein [29]. However, we believe that this cross reactivity does not have an impact on the current COVID 19 diagnosis because there are no new reported SARS-CoV outbreaks since 2004 [40].…”

“…It is worth noting that the immunosensor is easier to use compared with the other biosensors that needs an extraction step for the RNA [23,24]. Moreover, the LOD of the SARS-CoV-2 N protein immunosensor was lower than all the other reported electrochemical biosensors for the same protein [28][29][30][31] as shown in Table 1.…”

“…Molecular imprinted-based electrochemical biosensor was reported recently for the detection of N protein [28]. Two other magnetic-based electrochemical immunosensors were also developed recently for the detection of N protein in serum [29] and saliva [30]. We have recently reported a cottoncoated carbon nanofiber-based competitive biosensor for the detection of N protein [31].…”

Aptamers for Detection and Diagnostics (ADD): Can mobile systems process optical data from aptamer sensors to identify molecules indicating presence of SARS-CoV-2 virus? Should healthcare explore aptamers as drugs for prevention as well as its use as adjuvants with antibodies and vaccines?

“…: Example of smartphone tool using magnetic nanobeads in a microfludic immunosensor 339 which can detect SARS-CoV-2 nucleocapsid (N) protein in serum. The requirement for serum decreases its appeal as a tool for mass deployment.…”

Aptamers for Detection and Diagnostics (ADD): Can mobile systems process optical data from aptamer sensors to identify molecules indicating presence of SARS-CoV-2 virus? Should healthcare explore aptamers as drugs for prevention as well as its use as adjuvants with antibodies and vaccines?

“…Figure 23b: Example of smartphone tool using magnetic nanobeads in a microfludic immunosensor 339 which can detect SARS-CoV-2 nucleocapsid (N) protein in serum. The requirement for serum decreases its appeal as a tool for mass deployment.…”