Cost-Effective Modular Biosensor for SARS-CoV-2 and Influenza A Detection

Abstract

A modular, multi-purpose, and cost-effective electrochemical biosensor based on a five-stranded four-way junction (5S-4WJ) system was developed for SARS-CoV-2 (genes S and N) and Influenza A virus (gene M) detection. The 5S-4WJ structure consists of an electrode-immobilized universal stem-loop (USL) strand, two auxiliary DNA strands, and a universal methylene blue redox strand (UMeB). This design allows for the detection of specific nucleic acid sequences using square wave voltammetry (SWV). The sequence-specific auxiliary DNA strands (m and f) ensure selectivity of the biosensor for target recognition utilizing the same USL and UMeB components. An important feature of this biosensor is the ability to reuse the USL-modified electrodes to detect the same or alternative targets in new samples. This is accomplished by a simple procedure involving rinsing the electrodes with water to disrupt the 5S-4WJ structure and subsequent re-hybridization of the USL strand with the appropriate set of strands for a new analysis. The biosensor exhibited minimal loss in signal after rehybridization, demonstrating its potential as a viable multiplex assay for both current and future pathogens, with a low limit of quantification (LOQ) of as low as 17 pM.

Keywords: Influenza A; SARS-CoV-2; biosensor; four-way junction; microliter detection.

Figure 1

Scheme of the 5S-4WJ-based biosensor.

Figure 2

(A) Biosensor’s response towards varied concentrations (0, 1, 5, 10, 25, 40, 50, and 75 nM) of SARS-S target with a 10-min hybridization time on GDEs. Inset: Corresponding SWV for each of the concentrations tested. (B) Calibration curve for SARS N and InfA-M targets, both with a 10-min hybridization time on GDEs.

Figure 3

Regeneration and rehybridization of the USL probe for recognition of InfA-M and SARS-S. Square wave voltammetry response after hybridization with (A) m-InfA-M, f-InfA-M, InfA-M target and UMeB, (B) m-InfA-M, f-InfA-M, SARS-S target and UMeB, (C) m-SARS-S, f-SARS-S, SARS-S target and UMeB, (D) m-SARS-S, f-SARS-S, InfA-M target and UMeB on GDE. Black lines depict the baseline signal; blue lines depict the signal after hybridization or rehybridization of the USL-modified GDE with the indicated strands; red lines depict the signal after washing the 5S-4WJ components from the electrode (regeneration).

Figure 4

Response of the SARS-CoV-2 biosensor to the samples from NASBA (10% v/v). NTC: NASBA no-template control. Inset: dependence of the biosensor response on the logarithm of SARS-CoV-2 RNA concentration taken for the amplification step.