Impedimetric and amperometric genosensors for the highly sensitive quantification of SARS-CoV-2 nucleic acid using an avidin-functionalized multi-walled carbon nanotubes biocapture platform

Abstract

We report two novel genosensors for the quantification of SARS-CoV-2 nucleic acid using glassy carbon electrodes modified with a biocapture nanoplatform made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av) as a support of the biotinylated-DNA probes. One of the genosensors was based on impedimetric transduction offering a non-labelled and non-amplified detection of SARS-CoV-2 nucleic acid through the increment of [Fe(CN)₆]^3-/4- charge transfer resistance. This biosensor presented an excellent analytical performance, with a linear range of 1.0 × 10^-18 M - 1.0 × 10^-11 M, a sensitivity of (5.8 ± 0.6) x 10² Ω M^-1 (r² = 0.994), detection and quantification limits of 0.33 aM and 1.0 aM, respectively; and reproducibilities of 5.4% for 1.0 × 10^-15 M target using the same MWCNTs-Av-bDNAp nanoplatform, and 6.9% for 1.0 × 10^-15 M target using 3 different nanoplatforms. The other genosensor was based on a sandwich hybridization scheme and amperometric transduction using the streptavidin(Strep)-biotinylated horseradish peroxidase (bHRP)/hydrogen peroxide/hydroquinone (HQ) system. This genosensor allowed an extremely sensitive quantification of the SARS-CoV-2 nucleic acid, with a linear range of 1.0 × 10^-20 M - 1.0 × 10^-17 M, detection limit at zM level, and a reproducibility of 11% for genosensors prepared with the same MWCNTs-Av-bDNAp₁ nanoplatform. As a proof-of-concept, and considering the extremely high sensitivity, the genosensor was challenged with highly diluted samples obtained from SARS-CoV-2 RNA PCR amplification.

Keywords: Amperometric biosensor; Avidin; Carbon nanotubes; Impedimetric biosensor; Nanotechnology; SARS-CoV-2.

Fig. 1

Schematic representation of the steps followed during the construction of the impedimetric genosensor.

Fig. 2

(A) Nyquist plots obtained after the different steps during the construction of the impedimetric genosensor: a) GCE, b) GCE/MWCNTs-Av-bDNAp, c) GCE/MWCNTs-Av-DNAp/BSA, d) GCE/MWCNTs-Av-DNAp/BSA/target. Inset: equivalent circuit used to fit the impedance spectra. (B) Bars plot for the charge transfer resistances (R_ct) obtained from the Nyquist spectra shown in (A). Hybridization time: 60 min. Target concentration: 1.0 × 10⁻¹⁵ M. Redox marker: 1.0 × 10⁻³ M [Fe(CN)₆]³⁻/[Fe(CN)6]⁴⁻; Frequency range: 10 KHz to 10 mHz; Potential amplitude: 10 mV; Working potential: 0.200 V. Supporting electrolyte: 0.050 M phosphate buffer solution pH 7.40 with 0.500 M NaCl (PB–NaCl).

Fig. 3

(A) Nyquist plots obtained for GCE/MWCNTs-Av-bDNAp/BSA in the absence and presence of different target concentrations. (B) Calibration plot obtained from the results shown in (A). Other conditions as in Fig. 2.

Fig. 4

(A) Schematic representation of the steps followed during the construction of the sandwich hybridization-based amperometric genosensor. (B) Bars plot for the R_ct obtained after the different steps during the construction of the amperometric genosensor: a) GCE/MWCNTs-Av-bDNAp_1, b) GCE/MWCNTs-Av-bDNAp₁/BSA, c) GCE/MWCNTs-Av-bDNAp₁/BSA/target, d) GCE/MWCNTs-Av-bDNAp₁/BSA/target/bDNAp_2, e) GCE/MWCNTs-Av-bDNAp₁/BSA/target/bDNAp₂/Strep. Target concentration: 1.0 × 10⁻¹⁵ M. Hybridization time bDNAp₁/target and target/bDNAp₂: 60 min. Strep interaction time: 30 min. Inset: Nyquist plots obtained after the different steps. Other conditions as in Fig. 2.

Fig. 5

(A) Calibration plot obtained for the amperometric genosensor. Inset: amperometric response obtained in the absence and presence of different target concentrations. bHRP interaction time: 15 min. Working potential: -0.100 V Hydrogen peroxide concentration: 0.10 mM. HQ concentration: 0.50 mM. Other conditions as in Fig. 4 (B). Bars plot for the currents obtained from amperometric experiments using GCE/MWCNTs-Av-bDNAp₁/BSA under different conditions: a) in the presence of 1.0 × 10⁻¹⁷ M target; (b) in the absence of the target; (c) in the absence of bDNAp₂ for 1.0 × 10⁻¹⁷ M target; (d) in the absence of Strep for 1.0 × 10⁻¹⁷ M target; (e) in the presence of the 1.0 × 10⁻¹⁷ M scrambled sequence instead of the target; and (f) in the presence of non-biotinylated HRP for 1.0 × 10⁻¹⁷ M target. Other conditions as in Fig. 5A.

Fig. 6

Proof-of-concept of the potential analytical application of the genosensor. R_ct obtained for GCE/MWCNTs-Av-bDNAp/BSA before (a) and after (b, c) hybridization with PCR samples diluted with PB-NaCl 10¹⁰ (b) and 10⁴ (c) times. Other conditions as in Fig. 2.