Aptamer-Mediated Electrochemical Detection of SARS-CoV-2 Nucleocapsid Protein in Saliva

Abstract

Gold standard detection of SARS-CoV-2 by reverse transcription quantitative PCR (RT-qPCR) can achieve ultrasensitive viral detection down to a few RNA copies per sample. Yet, the lengthy detection and labor-intensive protocol limit its effectiveness in community screening. In view of this, a structural switching electrochemical aptamer-based biosensor (E-AB) targeting the SARS-CoV-2 nucleocapsid (N) protein was developed. Four N protein-targeting aptamers were characterized on an electrochemical cell configuration using square wave voltammetry (SWV). The sensor was investigated in an artificial saliva matrix optimizing the aptamer anchoring orientation, SWV interrogation frequency, and target incubation time. Rapid detection of the N protein was achieved within 5 min at a low nanomolar limit of detection (LOD) with high specificity. Specific N protein detection was also achieved in simulated positive saliva samples, demonstrating its feasibility for saliva-based rapid diagnosis. Further research will incorporate novel signal amplification strategies to improve sensitivity for early diagnosis.

Keywords: SARS-CoV-2 nucleocapsid protein; electrochemical sensor; structural switching aptamer.

Figure 1

Design and mechanism of the E-AB system. SARS-CoV-2 nucleocapsid protein binding induces conformational change in the aptamers immobilized on working electrode. This results in an alteration of the distance between the MB reporter and the gold surface and leads to an increase in the current output of the system, which corresponds to the concentration of the nucleocapsid proteins in the sample.

Figure 2

Comparison of aptamer anchoring geometry. Four selected N aptamers were decorated onto gold via either 5′ or 3′ Au-S self-assembly reaction. Blank and 50 nM target N protein in 30% artificial saliva/PBS was applied. Binding signals were measured by SWV. *, p < 0.05, **, p < 0.01.

Figure 3

Optimization of E-AB target incubation time. Aptamers (A) A15, (B) A58, (C) A48, and (D) A61 sensors were incubated in 50 nM of N protein. The current outputs were continuously monitored from 0 to 12 min by SWV every 15 s.

Figure 4

N protein concentration responses of (A) A15, (B) A58, (C) A48, and (D) A61 E-ABs. Measurements conducted using 5′ aptamer-conjugated E-ABs at 200 Hz in 30% artificial saliva/PBS. Linear regression model was used to fit the dose responses. LOD was defined as 3.3*(Sy/S), where Sy is standard error of y-intercept and S is slope of regression.

Figure 5

E-AB specificity in artificial saliva and human saliva matrices. (A) E-ABs decorated with aptamers A15, A58, A48, and A61 and random sequence were tested against 100 nM of target N protein and non-target S protein or BSA. Target protein-specific and sequence-specific detection can be concluded. (B) A15 E-AB was tested against target N protein and non-target S protein and BSA in three different confirmed COVID-19-negative saliva samples. ***, p < 0.001.