Aptamer-based electrochemical biosensor for rapid detection of SARS-CoV-2: Nanoscale electrode-aptamer-SARS-CoV-2 imaging by photo-induced force microscopy

Abstract

Rapid, mass diagnosis of the coronavirus disease 2019 (COVID-19) is critical to stop the ongoing infection spread. The two standard screening methods to confirm the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are polymerase chain reaction (PCR), through the RNA of the virus, and serology by detecting antibodies produced as a response to the viral infection. However, given the detection complexity, cost and relatively long analysis times of these techniques, novel technologies are urgently needed. Here, we report an aptamer-based biosensor developed on a screen-printed carbon electrode platform for rapid, sensitive, and user-friendly detection of SARS-CoV-2. The aptasensor relies on an aptamer targeting the receptor-binding domain (RBD) in the spike protein (S-protein) of the SARS-CoV-2. The aptamer immobilization on gold nanoparticles, and the presence of S-protein in the aptamer-target complex, investigated for the first time by photo-induced force microscopy mapping between 770 and 1910 cm^-1 of the electromagnetic spectrum, revealed abundant S-protein homogeneously distributed on the sensing probe. The detection of SARS-CoV-2 S-protein was achieved by electrochemical impedance spectroscopy after 40 min incubation with several analyte concentrations, yielding a limit of detection of 1.30 pM (66 pg/mL). Moreover, the aptasensor was successfully applied for the detection of a SARS-CoV-2 pseudovirus, thus suggesting it is a promising tool for the diagnosis of COVID-19.

Keywords: Aptasensor; Detection; Photo-induced force microscopy; S-protein; SARS-CoV-2.

Scheme 1

Stepwise fabrication of aptasensor for SARS-CoV-2 S-protein detection.

Fig. 1

a) CVs (first and second cycle) of Au(lII) reduction at a SPCE recorded in 5 mM HAuCl₄ + 0.1 M NaNO₃ (50 mV s⁻¹ scan rate), b) Current transient and charge consumed recorded during electrodeposition of AuNPs on a SPCE from a 5 mM HAuCl₄ + 0.1 M NaNO₃ solution, c) electrochemical profile of AuNPs/SPCE recorded in 0.5 M H₂SO₄ (50 mV s⁻¹ scan rate), and d) three-dimensional AFM image of the AuNPs/SPCE.

Fig. 2

a) CV and b) EIS measurements for every step of the aptasensor fabrication, recorded in PBS solution containing 5 mM [Fe(CN)₆]^3-/4- (c–e) AFM and PiFM characterization of the modified electrode after each modification step. c) Three-dimensional topography and PiFM screening at ~1400 cm⁻¹ representations of the AuNPs-, Aptamer/AuNPs- and S-protein/Aptamer/AuNPs-modified SPCEs. According to the PiF-IR spectra (Fig. 2d) and targeting selected peak shifts for best chemical contrast, the common wavenumbers at (~1050, ~1390 cm⁻¹ and 1680–1730 cm⁻¹) were selected for PiFM mapping. d) Representative PiF-IR spectra acquired on the Aptamer/AuNPs- and S-protein/Aptamer/AuNPs-modified SPCEs, respectively in blue and green. Highlighted regions are mutual resonance peaks observed in both samples e) AFM and PiFM 500 × 500 nm² micrographs of the AuNPs-, Aptamer/AuNPs-and S-protein/Aptamer/AuNPs-modified SPCEs at the highlighted region wavenumbers in d), ~1050, ~1390 and ~1680-1730 cm⁻¹. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

a) Nyquist plots of the aptasensor response towards different concentrations of S-protein, recorded in PBS solution containing 5 mM [Fe(CN)₆]^3-/4-, solid lines correspond to the experimental data, while symbols correspond to the EIS data fitting, and b) calibration curve of the aptasensor with logarithmic S-protein concentration.

Fig. 4

a) Selectivity of the aptasensor studied with the S-proteins of MERS-CoV, SARS-CoV and SARS-CoV-2, and b) Impedimetric response obtained with 50 nM SARS-CoV-2 S-protein to evaluate the stability of the aptasensor after 3 weeks storing in BB.

Fig. 5

Impedimetric response of the aptasensor towards the HIV_NL4-3Env-luc and the HIV_NL4-3Env-luc + S-protein pseudovirus. The inset is the percentage change in charge transfer resistance calculated from the EIS data.