A nanoscale genosensor for early detection of COVID-19 by voltammetric determination of RNA-dependent RNA polymerase (RdRP) sequence of SARS-CoV-2 virus

Abstract

A voltammetric genosensor has been developed for the early diagnosis of COVID-19 by determination of RNA-dependent RNA polymerase (RdRP) sequence as a specific target of novel coronavirus. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) uses an RdRP for the replication of its genome and the transcription of its genes. Here, the silver ions (Ag⁺) in the hexathia-18-crown-6 (HT18C6) were used for the first time as a redox probe. Then, the HT18C6(Ag) incorporated carbon paste electrode (CPE) was further modified with chitosan and PAMAM dendrimer-coated silicon quantum dots (SiQDs@PAMAM) for immobilization of probe sequences (aminated oligonucleotides). The current intensity of differential pulse voltammetry using the redox probe was found to decrease with increasing the concentration of target sequence. Based on such signal-off trend, the proposed genosensor exhibited a good linear response to SARS-CoV-2 RdRP in the concentration range 1.0 pM-8.0 nM with a regression equation I (μA) = - 6.555 log [RdRP sequence] (pM) + 32.676 (R² = 0.995) and a limit of detection (LOD) of 0.3 pM. The standard addition method with different spike concentrations of RdRP sequence in human sputum samples showed a good recovery for real sample analysis (> 95%). Therefore, the developed voltammetric genosensor can be used to determine SARS-CoV-2 RdRP sequence in sputum samples. PAMAM-functionalized SiQDs were used as a versatile electrochemical platform for the SARS-CoV-2 RdRP detection based on a signal off sensing strategy. In this study, for the first time, the silver ions (Ag⁺) in the hexathia-18-crown-6 carrier were applied as an electrochemical probe.

Keywords: Genosensor; PAMAM@SiQDs; SARS-CoV-2 RdRP sequence.

PAMAM-functionalized SiQDs were used as a versatile electrochemical platform for the SARS-CoV-2 RdRP detection based on a signal off sensing strategy. In this study, for the first time, the silver ions (Ag⁺) in the hexathia-18-crown-6 carrier were applied as an electrochemical probe.

Fig. 1

a TEM image, b EDX spectrum, c UV-vis and fluorescence spectra of SiQDs, and d TEM image of SiQDs@PAMAM; the magnification of TEM images was adjusted in 100 KX

Fig. 2

a EDX spectrum of CPE-HT18C6(Ag); SEM images of b CPE-HT18C6(Ag) and c CPE-HT18C6(Ag)/chitosan with magnification of 2.00 KX

Fig. 3

Schematic illustration of fabrication process of electrochemical genosensor

Fig. 4

CVs of CPE-HT18C6(Ag) (a), CPE-HT18C6(Ag)/chitosan (b), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM (c), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence (d), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence/SARS-CoV-2 RdRP sequence (1 pM) (e) in A 0.1 M PBS (pH = 7.2) and B 0.1 M KCl containing 5 mM Fe(CN)₆^3−/4−. C Nyquist plots of CPE-HT18C6(Ag) (a), CPE-HT18C6(Ag)/chitosan (b), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM (c), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence (d), CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence/SARS-CoV-2 RdRP sequence (1 pM) (e) in 0.1 M KCl containing 5 mM Fe(CN)₆^3−/4 −

Fig. 5

a DPVs of CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence in the presence of different concentrations of SARS-CoV-2 RdRP sequence (0, 1 pM, 10 pM, 100 pM, 1000 pM, 5000 pM, 8000 pM) in 0.1 M PBS of pH 7.2; b Calibration curve of I_p vs. log C_RdRP

Fig. 6

A DPVs of CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence in (a) PBS of pH 7.2, (b) E gene (50 pM), (c) SARS RdRP gene (50 pM), and (d) SARS-CoV-2 RdRP gene (10 pM). B Bar charts of DPV responses of CPE-HT18C6(Ag)/chitosan/SiQDs@PAMAM/probe sequence in PBS of pH 7.2, SARS-CoV-2 RdRP gene (10 pM), and the mixtures of SARS-COV-2 RdRP (10 pM) and other interferent genes (50 pM)