SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex

Abstract

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 μA∗mL/μg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient.

Keywords: ACE2; Coronavirus; Immunosensor; Magnetic beads; SARS-CoV-2; Spike protein.

Graphical abstract

Scheme 1

Conceptual schematic of the immunosensor design based on the Spike-ACE-2 complex. A) Shandwich immunosensor assembly on the magnetic bead-based platform and B) enzyme-amplified electrochemical reaction and chronoamperometric signal readout [49].

Fig. 1

Immunosensor response with increasing concentrations of spike protein amplified with streptavidin-HRP. A) Electrochemical and B) spectrophotometric response with 0, 0.1, 0.6, 1, 1.5 and 2 μg/mL spike protein, C) Correlation between electrochemical and optical reading.

Fig. 2

A) Immunosensor electrochemical response with increasing concentrations of 0, 0.1, 0.3, 0.5, 0.8 and 1 μg/mL of the spike protein amplified with streptavidin-HRP (poly) 80 and B) Corresponding calibration curve.

Fig. 3

Immunosensor electrochemical response with increasing concentrations of 0, 1, 10, 10², 10³, 10⁴, and 10⁶ copies/mL pseudovirions amplified with streptavidin-HRP (poly) 80 and B) Corresponding calibration curve.

Fig. 4

Immunosensor specificity when detecting 1 μg/mL spike, RBD and β-1,4-GALT-5 proteins and 1 × 10⁵ copies/mL MERS, SARS-CoV-1, SARS-CoV-2 and VSV pseudovirion supernatants. Statistically significant differences (a) with respect to the negative control and (b) with respect to the commercial spike protein, ∗∗∗(p < 0.001), ∗∗(p < 0.01), ∗(p < 0.05) and – (p > 0,05).

Fig. 5

Immunosensor response of different lysed patient samples compared to a commercial spike protein-positive control and a negative control without the target protein. Cyan, purple, green and blue columns correspond to negative samples and high, medium and low C.T positive samples. Statistically significant differences (a) with respect to the negative control and (b) with respect to the commercial spike protein, ∗∗∗(p < 0.001), ∗∗(p< 0.01), ∗(p < 0.05) and – (p> 0,05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)