A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization

Abstract

To satisfy the need to develop highly sensitive methods for detecting the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and further enhance detection efficiency and capability, a new method was created for detecting SARS-CoV-2 of the open reading frames 1ab (ORF1ab) target gene by a electrochemiluminescence (ECL) biosensor based on dual-probe hybridization through the use of a detection model of “magnetic capture probes—targeted nucleic acids—Ru(bpy)32+ labeled signal probes”. The detection model used magnetic particles coupled with a biotin-labeled complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab target gene as the magnetic capture probes and Ru(bpy)32+ labeled amino modified another complementary nucleic acid sequence as the signal probes, which combined the advantages of the highly specific dual-probe hybridization and highly sensitive ECL biosensor technology. In the range of 0.1 fM~10 µM, the method made possible rapid and sensitive detection of the ORF1ab gene of the SARS-CoV-2 within 30 min, and the limit of detection (LOD) was 0.1 fM. The method can also meet the analytical requirements for simulated samples such as saliva and urine with the definite advantages of a simple operation without nucleic acid amplification, high sensitivity, reasonable reproducibility, and anti-interference solid abilities, expounding a new way for efficient and sensitive detection of SARS-CoV-2.

A Study of the Detection of SARS-CoV-2 by the Use of Electrochemiluminescent Biosensor Based on Asymmetric Polymerase Chain Reaction Amplification Strategy

“…Consequently, per 200 µg of magnetic particles, the optimal addition amount of biotin-probes is 150 pmol. At this point, the immobilized amount of biotin-probes per 200 µg of magnetic particles is 71.32 pmol [18].…”

“…Consequently, per \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$200 ~\mu \text{g}$ \end{document} of magnetic particles, the optimal addition amount of biotin-probes is 150 pmol. At this point, the immobilized amount of biotin-probes per \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$200 ~\mu \text{g}$ \end{document} of magnetic particles is 71.32 pmol [18] .…”

“…Curve b is the spectrum of amino probes. Curve c is the spectrum of Ru(bpy) 2+ 3 -labeled signal probes [18]. with the ORF1ab target gene addition.…”

“…In the same conditions, utilizing the magnetic capture probes and the luminescent probe designed in this study, a method was constructed for detecting SARS-CoV-2 by ECL biosensor based on dual-probes hybridization using a detection model of magnetic capture probes-targeted nucleic acids-Ru(bpy) 2+ 3 -labeled luminescent probes, for which LOD was 60.2 copies/µL (100 aM) [18]. In our study, the sensitivity of the ECL biosensor based on asymmetric PCR amplification for SARS-CoV-2 ORF1ab gene detection was 60.2 times higher than the ECL biosensor based on dual-probes hybridization one.…”

A visual detection strategy for SARS-CoV-2 based on dual targets-triggering DNA walker

“… Methods Strategy LOD Ref. Electrochemical boron nitride quantum dots / flower-like gold nanostructures signal amplification 0.48 aM [39] Fluorescent Exo III and DNAzyme-mediated signal amplification 3.3 fM [14] Colorimetric thiol-modified antisense oligonucleotide-capped AuNPs 0.18 ng μL -1 [40] Electrochemical dual-probe hybridization based on magnetic capture probes and target 0.1 fM [41] Electrochemical based on gold nanomaterials electrodes and signal of [Ru(bpy) 3 ] 2+ /carbon nanodots 514 aM [42] Electrochemical morpholino-modified graphene field-effect transistor nanosensor 0.37 fM [43] Electrochemical bipedal DNA walker amplification strategy 7.8 aM [16] Electrochemical DNA Walker based on Au@Ti3C2@PEI-Ru(dcbpy)32+ nanocomposite 0.21 fM [15] Colorimetric DNA aptamers 20 pM [44] Colorimetric and Data analysis based on Smartphone SCGS/DNA walker combined with enzyme-catalyzed amplification 1.19 pM This work …”