Impedimetric Detection Based on Label-Free Immunoassay Developed for Targeting Spike S1 Protein of SARS-CoV-2

Abstract

After the COVID-19 pandemic started all over the world, great importance was placed on the development of sensitive and selective bioanalytical assays for the rapid detection of the highly pathogenic SARS-CoV-2 virus causing COVID-19 disease. In this present work, an impedimetric immunosensor was developed and applied for rapid, reliable, sensitive and selective detection of the SARS-CoV-2 S1 protein. To detect the SARS-CoV-2 virus, targeting of the spike S1 protein was achieved herein by using S1 protein-specific capture antibody (Cab-S1) immobilized screen-printed electrode (SPE) in combination with the electrochemical impedance spectroscopy (EIS) technique. With the impedimetric immunosensor, the detection limit for S1 protein in buffer medium was found to be 0.23 ng/mL (equal to 23.92 amol in 8 µL sample) in the linear concentration range of S1 protein from 0.5 to 10 ng/mL. In the artificial saliva medium, it was found to be 0.09 ng/mL (equals to 9.36 amol in 8 µL sample) in the linear concentration range of S1 protein between 0.1 and 1 ng/mL. The selectivity of the impedimetric immunosensor toward S1 protein was tested against influenza hemagglutinin antigen (HA) in the buffer medium as well as in artificial saliva.

Keywords: COVID-19; SARS-CoV-2 S1 protein; electrochemical immunosensors; electrochemical impedance spectroscopy.

Figure 1

Development of label-free immunosensor with its application for impedimetric detection of spike S1 protein of SARS-CoV-2.

Figure 2

(A) Nyquist diagrams showing the data obtained by impedimetric immunosensor for the detection of S1 protein in its concentrations varying from 0.5 to 10 ng/mL prepared in buffer medium. (B) Calibration curve obtained by impedimetric detection of spike S1 protein of SARS-CoV-2 (n = 6).

Figure 3

Histograms presenting the data obtained in the selectivity study performed in different concentrations of S1 protein or HA. Gray columns represent full procedure in the presence of spike S1 protein, and green-striped columns represent full procedure in the presence of HA (n = 3).

Figure 4

(A) Nyquist diagrams showing the response of the label-free impedimetric immunosensor at increasing concentrations of S1 protein in the range of 0–1 ng/mL prepared in diluted artificial saliva (1:20). (B) Calibration curve presenting the data for the impedimetric determination of spike S1 protein of SARS-CoV-2 in diluted artificial saliva (1:20) (n = 3).

Figure 5

Histograms presenting the data obtained in the selectivity study performed on different concentrations of S1 protein or HA prepared in diluted artificial saliva (1:20). Gray columns represent full procedure in the presence of spike S1 protein, and green-striped columns represent full procedure in the presence of HA (n = 3).

Figure 6

Histograms presenting the data obtained in the selectivity study performed with 1 ng/mL S1 protein, 1 ng/mL HA and the mixture sample containing 1 ng/mL S1 protein and 1 ng/mL HA prepared in diluted artificial saliva (1:20) (n = 3).