An electrochemical membrane-based aptasensor for detection of severe acute respiratory syndrome coronavirus-2 receptor-binding domain

Abstract

Herein, we report an electrochemical membrane-based aptasensor for the determination of the SARS-CoV-2 receptor-binding domain (SARS-CoV-2-RBD). For this purpose, the nanoporous anodic aluminium oxide membrane (NPAOM) was first fabricated electrochemically. The NPAOM was then functionalized with 3-mercaptopropyl trimethoxysilane (NPAOM-Si-SH). After that, the NPAOM-Si-SH was decorated with gold nanoparticles by using gold ion and sodium borohydride. The NPAOM-Si-S-Au_nano was then attached to the surface of the working electrode of a laser-engraved graphene electrode (LEGE). Subsequently, the LEGE/NPAOM-Si-S-Au_nano was fixed inside a flow cell that was made by using a three-dimensional (3D) printer, and then thiolated aptamer was transferred into the flow cell using a pump. The electrochemical behavior of the LEGE/NPAOM-Si-S-Au_nano-Aptamer was studied using square wave voltammetry (SWV) in the presence of potassium ferrocyanide as a redox probe. The response of the LEGE/NPAOM-Si-S-Au_nano-Aptamer to the different concentrations of the SARS-CoV-2-RBD in human saliva sample was investigated in the concentration range of 2.5-40.0 ng/mL. The limit of the detection was found to be 0.8 ng/mL. The LEGE/NPAOM-Si-S-Au_nano-Aptamer showed good selectivity to 5.0 ng/mL of SARS-CoV-2-RBD in the presence of five times of the interfering agents like hemagglutinin and neuraminidase as the influenza A virus major surface glycoproteins.

Keywords: 3-D printed flow-cell; Electrochemical aptasensor; Laser-engraved graphene electrode; Nanoporous anodic aluminium oxide membrane decorated with gold nanoparticles; SARS-CoV-2-RBD.

Graphical abstract

Fig. 1

Schematic illustration for the fabrication of the membrane-based aptasensor.

Fig. 2

SEM images of the NPAOM (A-B) and NPAOM-Au_nano (C-E).

Fig. 3

Fourier transform infrared spectrum of NPAOM (a) NPAOM-Si-S-Au_nano-Aptamer (b) (A). Raman spectrum of NPAOM-Si-S-Au_nano-Aptamer (B).

Fig. 4

SWVs of the LEGE/NPAOM-Si-S-Au_nano (a), LEGE/NPAOM-Si-S-Au_nano-Aptamer (b), and LEGE/NPAOM-Si-S-Au_nano-Aptamer/SARS-CoV-2 RBD (c) in 0.1 M PBS containing 1 mM Fe(CN)₆⁴⁻ solution.

Fig. 5

Effect of the immobilized aptamer (A) and the incubation time (B) on the response of LEGE/NPAAO-Si-S-Au_nano-Aptamer to 40.0 ng/mL SARS-CoV-2 RBD in 0.1 M PBS containing 1.0 mM Fe(CN)₆⁴⁻ solution.

Fig. 6

SWV of LEGE/NPAOM-Si-S-Au_nano-Aptamer at the optimum operating conditions for different amounts of SARS-CoV-2 RBD in a 1 mM Fe(CN)₆⁴⁻ solution (pH 7.4) (A). The corresponding calibration plots of SWV response toward SARS-CoV-2 RBD (2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 ng/mL) (B). The error bars were obtained by using four different aptasensors. The selectivity of LEGE/NPAOM-Si-S-Au_nano-Aptamer to 5.0 ng/mL SARS-CoV-2 RBD in the absence (black curve) and presence of 5 times excess of HA (red curve) and NA (green curve) (C). The stability of the signal of the LEGE/NPAOM-Si-S-Au_nano-Aptamer to 5.0 ng/mL SARS-CoV-2 RBD before (a) and after (b) 3 weeks (D).