Impedimetric Sensor for SARS-CoV-2 Spike Protein Detection: Performance Assessment with an ACE2 Peptide-Mimic/Graphite Interface

Abstract

The COVID-19 pandemic has prompted the need for the development of new biosensors for SARS-CoV-2 detection. Particularly, systems with qualities such as sensitivity, fast detection, appropriate to large-scale analysis, and applicable in situ, avoiding using specific materials or personnel to undergo the test, are highly desirable. In this regard, developing an electrochemical biosensor based on peptides derived from the angiotensin-converting enzyme receptor 2 (ACE2) is a possible answer. To this end, an impedimetric detector was developed based on a graphite electrode surface modified with an ACE2 peptide-mimic. This sensor enables accurate quantification of recombinant 2019-nCoV spike RBD protein (used as a model analyte) within a linear detection range of 0.167-0.994 ng mL^-1, providing a reliable method for detecting SARS-CoV-2. The observed sensitivity was further demonstrated by molecular dynamics that established the high affinity and specificity of the peptide to the protein. Unlike other impedimetric sensors, the herein presented system can detect impedance in a single frequency, allowing a measure as fast as 3 min to complete the analysis and achieving a detection limit of 45.08 pg mL^-1. Thus, the proposed peptide-based electrochemical biosensor offers fast results with adequate sensitivity, opening a path to new developments concerning other viruses of interest.

Keywords: ACE2 peptide-mimic; biosensors; graphite surface; impedimetric detection; spike protein detection.

Figure 1

Simplified workflow of the (a) fabrication of the biosensor based on ACE2 peptide-mimetic and (b) recombinant SARS-CoV-2 spike RBD protein detection. The modification of the graphite surface is achieved by incorporating -COOH residues, which serve as anchors for the immobilization of the ACE2 peptide-mimetic (ACE2p). The recombinant SARS-CoV-2 RBD protein is detected by measuring the system’s total impedance using [Fe(CN)₆]^−3/−4 as a redox probe.

Figure 2

Selected conformers for the coupling simulation study.

Figure 3

Representation of the binding site and the complexes formed between ACE2-RBD, obtained from the crystallographic structure (PDB-ID: 6CS2), and the different conformers of the ACE2 peptide-mimic complex generated through docking simulation using HADDOCK. (A) ACE2-RBD complex, (B) RBD–Conformer 1, (C) RBD–Conformer 2, (D) RBD–Conformer 3, and (E) RBD–Conformer 4.

Figure 4

SEM/EDS analysis of three graphite electrodes: (a) blank graphite electrode (graphite), (b) electrografted graphite electrode (EG–graphite), and (c) peptide-modified graphite electrode (ACE2p–graphite). The crosses in each SEM image indicate the regions where the composition analysis was performed. The corresponding atomic concentrations of carbon, oxygen, and nitrogen are shown below each image. Scale bars represent 50 µm.

Figure 5

Comparison of electrode surfaces via AFM. (a) Blank graphite electrode (graphite), (b) electrografted graphite electrode (EG–graphite), and (c) ACE2p-modified graphite electrode (ACE2p–graphite).

Figure 6

Cyclic voltammogram of [Fe(CN)₆]^4−/3− 5 mM in PBS 1× pH 7.4 using graphite (black), EG-graphite (blue), and ACE2p-graphite (red) as the working electrode.

Figure 7

(a) Bode graph of a series of solutions with increasing concentration of recombinant 2019-nCoV spike RBD protein. (b) Variation of total impedance with respect to the concentration of recombinant 2019-nCoV spike RBD protein.