Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

Nadiah Ibrahim; Kok Beng Gan; Nurul Yuziana Mohd Yusof; Choo Ta Goh; Niranjana Krupa B; Ling Ling Tan

doi:10.1016/j.talanta.2024.125916

Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

Talanta. 2024 Mar 18:274:125916. doi: 10.1016/j.talanta.2024.125916. Online ahead of print.

Authors

Nadiah Ibrahim¹, Kok Beng Gan², Nurul Yuziana Mohd Yusof³, Choo Ta Goh⁴, Niranjana Krupa B⁵, Ling Ling Tan⁶

Affiliations

¹ Southeast Asia Disaster Prevention Research Initiative (SEADPRI), Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia. Electronic address: p110052@siswa.ukm.edu.my.
² Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia. Electronic address: kbgan@ukm.edu.my.
³ Department of Earth Sciences and Environment, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia. Electronic address: yuziana@ukm.edu.my.
⁴ Southeast Asia Disaster Prevention Research Initiative (SEADPRI), Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia. Electronic address: gohchoota@ukm.edu.my.
⁵ Department of Electronics and Communication Engineering, PES University, Bengaluru-85, Karnataka, India. Electronic address: bnkrupa@pes.edu.
⁶ Southeast Asia Disaster Prevention Research Initiative (SEADPRI), Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia. Electronic address: lingling@ukm.edu.my.

PMID: 38547835
DOI: 10.1016/j.talanta.2024.125916

Abstract

In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R² = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

Keywords: COVID-19; Differential pulse voltammetry; G-quadruplex; Genosensor; Methylene blue; qRT-PCR.