A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein

Abstract

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (K_D) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

Keywords: COVID-19; DNA aptamer; DNA nanotechnology; electrochemical sensors; single-walled carbon nanotubes.

Figure 1

(a) Schematic illustration of the working principle of the aptasensor based on the conformational change in a redox-tagged SARS-CoV-2 aptamer upon interaction with the target S1 protein. A suppression of the output current is observed in the presence of S1 protein because of the structural rearrangement in the aptamer folding that moves the redox reporter away from the electrode surface. (b) Secondary structure of the AttoMB2-modified SARS-CoV-2 aptamer used in this work, based on a recently discovered aptamer sequence.

Figure 2

(a) DPV voltammograms obtained in the presence of S1 SARS-CoV-2 protein in the concentration range 0.3–300 nM. (b) Binding curve based on a Langmuir-type equation describing the response current as a function of the S1 protein concentration. Highlighted is the concentration range in which response is linear. (c) Calibration curve obtained by linear fit of the response current values in the 20–100 nM S1 protein concentration range (in all the figures, data are reported as mean value ± SD, n = 3).

Figure 3

(a) Schematic illustration of the aptasensor behavior when pre-treating the target S1 protein with the same SARS-CoV-2 aptamer, lacking the redox reporter, as a competitor in solution. (b) DPV voltammograms in the absence of the target protein (only aptamer, dark blue line), in the presence of 100 nM S1 protein pre-incubated with an excess of competitor aptamer (+ S1 protein + competitor, green line), and in the presence of the S1 protein at 100 nM concentration (+ S1 protein, light blue line). (c) Relative signal suppression obtained when measuring the amperometric current in the presence of S1 protein 100 nM (light blue bar) and in the presence of the same protein incubated with the aptamer competitor (white bar). (d) Specificity of the sensor evaluated by using different aptamer sequences immobilized onto the electrode surface (SARS-CoV-2, PDGF, and thrombin aptamer) in the presence of S1 protein at 100 nM concentration in PBS (left panel), and by using different proteins at 100 nM concentration in PBS (S1 SARS-CoV-2, S1 MERS and H1N1) when the SARS-CoV-2 aptamer is immobilized onto the electrode surface (right panel) (in all the figures data are reported as mean value ± SD, n = 3).

Figure 4

(a) DPV voltammogram obtained in the presence of target S1 protein in VTM at 50 and 100 nM. (b) Histograms showing the sensor performance, expressed as signal suppression %, when the S1 protein is dissolved in PBS (blue bars) or VTM (red bars) (mean value ± SD, n = 3).