Rational engineering the DNA tetrahedrons of dual wavelength ratiometric electrochemiluminescence biosensor for high efficient detection of SARS-CoV-2 RdRp gene by using entropy-driven and bipedal DNA walker amplification strategy

Abstract

Fast and effective detection of epidemics is the key to preventing the spread of diseases. In this work, we constructed a dual-wavelength ratiometric electrochemiluminescence (ECL) biosensor based on entropy-driven and bipedal DNA walker cycle amplification strategies for detection of the RNA-dependent RNA polymerase (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The entropy-driven cyclic amplification reaction was started by the SARS-CoV-2 RdRp gene to generate a bandage. The bandage could combine with two other single-stranded S1 and S2 to form a bipedal DNA walker to create the following cycle reaction. After the bipedal DNA walker completed the walking process, the hairpin structures at the top of the DNA tetrahedrons (TDNAs) were removed. Subsequently, the PEI-Ru@Ti₃C₂@AuNPs-S7 probes were used to combine with the excised hairpin part of TDNAs on the surface of Au-g-C₃N₄, and the signal change was realized employing electrochemiluminescence resonance energy transfer (ECL-RET). By combining entropy-driven and DNA walker cycle amplification strategy, the ratiometric ECL biosensor exhibited a limit of detection (LOD) as low as 7.8 aM for the SARS-CoV-2 RdRp gene. As a result, detecting the SARS-CoV-2 RdRp gene in human serum still possessed high recovery so that the dual-wavelength ratiometer biosensor could be used in early clinical diagnosis.

Keywords: DNA tetrahedrons; Electrochemiluminescence biosensor; Entropy-driven; Ratiometric; SARS-CoV-2; bipedal DNA walker.

Scheme 1

Schematic illustration of the entropy-driven and bipedal DNA walker amplification strategies based DNA tetrahedral ratiometric ECL biosensor for the assay SARS-CoV-2 RdRp gene.

Fig. 1

(A) UV–Vis absorption spectrum of PEI-Ru@Ti₃C₂@AuNPs (gray curve) and ECL spectrum of Au-g-C₃N₄ (red curve), ECL curves were obtained through a series of filters, measured in 0.1 M PBS (pH = 7.4) containing 0.1 M S₂O₈^2-. (B) ECL spectrum of GCE/Au-g-C₃N₄ (curve a) and GCE/PEI-Ru@Ti₃C₂@AuNPs (curve b), scanned at −1.5–0 V. (C) ECL signal intensity of (a) GCE/Au-g-C₃N₄/TDNAs/MCH, (b) GCE/Au-g-C₃N₄/TDNAs/MCH treated with 10 pM of SARS CoV-2 RdRp and further incubated with PEI-Ru@Ti₃C₂@AuNPs-S7. (D) Ratiometric ECL biosensor at different concentrations (0 aM, 10 aM, 10 fM, and 10 pM) of SARS CoV-2 RdRp was tested using a series of filters spaced 20 nm apart, measured in 0.1 M PBS (pH = 7.4) containing 0.1 M S₂O₈^2-. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

(A) TEM characterization of Au-g-C₃N₄, inset shows g-C₃N₄. (B) EDX spectrum of Au-g-C₃N₄, inset shows EDX of g-C₃N₄. (C) UV–vis absorption spectrum of g-C₃N₄ (curve a) and Au-g-C₃N₄ (curve b). (D) ECL intensity curves of g-C₃N₄ (curve a) and Au-g-C₃N₄ (curve b), cyclic voltammetry curves of g-C₃N₄ (curve a’) and Au-g-C₃N₄ (curve b’), modified on GCE tested in 0.1 M PBS (pH = 7.4) containing 0.1 M S₂O₈^2-.

Fig. 3

(A) TEM characterization of Ti₃C₂. (B) TEM characterization of PEI-Ru@Ti₃C₂@AuNPs. (C) The corresponding size distribution diagram of (B). (D) The EDX spectrum of PEI-Ru@Ti₃C₂@AuNPs.

Fig. 4

Polyacrylamide gel electrophoresis analysis of (A) TDNAs construction process. Lane 1: S3, Lane 2: S3-S4, Lane 3: S3-S4-S5, Lane 4: S3-S4-S5-S6. (B) Entropy-driven cyclic amplification reaction, Lane 1: Scaffold, Lane 2: Blocker, Lane 3: Bandage, Lane 4: Substrate: Scaffold + Blocker + Bandage, Lane 5: Substrate + SARS-CoV-2 RdRp, Lane 6: Substrate + SARS-CoV-2 RdRp + Fuel. (C) Bipedal DNA walker, Lane 1: S1 probe, Lane 2: S2 probe, Lane 3: S1 + S2, Lane 4: S1 + S2 + Bandage.

Fig. 5

Characterization of the stepwise construction process of the biosensor with CV (A), EIS (B) and ECL (C). (a) bare GCE, (b) GCE/Au-g-C₃N₄, (c) GCE/Au-g-C₃N₄/TDNAs, (d) GCE/Au-g-C₃N₄/TDNAs/MCH, (e) GCE/Au-g-C₃N₄/TDNAs/MCH treated with SARS CoV-2 RdRp and Nb.BbvCI, (f) Futher incubation with PEI-Ru@Ti₃C₂@AuNPs-S7. The inset in Fig. 5B was the modified Randles circuit to simulate the electrochemical process during electrode modification.

Fig. 6

(A) Relationship between the ECL intensity of (a) Au-g-C₃N₄ at 460 nm and (b) PEI-Ru@Ti₃C₂@AuNPs at 620 nm and the logarithmic value of SARS-CoV-2 RdRp concentrations (10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM). (B) The calibration curve for SARS-CoV-2 RdRp. (C) Stability of the ECL biosensor with 16 continuous measurements at 460 nm (left) and 620 nm (right), respectively, with a SARS-COV-2 RdRp concentration of 10 pM. (D) Selectivity of ratiometric ECL biosensor at (a) 1 pM for SARS-CoV-2 RdRp, (b) 100 pM for SARS-CoV RdRp, (c) 100 pM for random DNA, (d) blank solution. The ECL measurement used a series of filters spaced 20 nm apart, measured in 0.1 M PBS (pH = 7.4) containing 0.1 M S₂O₈^2-.