RNA-responsive optode DNA slide probe: A study to unravel the label- and amplification-free optosensing mechanisms of emerging and pathogenic RNA virus.

A novel optical signal read-out avenue based on dynamic surface transformation at the optode DNA slide probe immobilized with methylene blue (Mb)-binding human telomeric G-quadruplex DNA for label-free optosensing of viral RNA using synthetic N2 gene SARS-CoV-2 as the model target RNA. The high-affinity chromophoric Mb binding ligand towards G-quadruplex DNA inserted with receptor loop, formed a strong binding by end-stacking with π system of the guanine quartet in the compaction hybrid-type of mixed parallel/antiparallel G-quadruplex-Mb structure, with maximum optical absorption transduction attained at 664 nm. Hybridization of the complementary receptor loop with target viral gene induced relaxation to the G-quadruplex conformation, transforming it into duplex structure, which significantly amplified its characteristic absorbance signal, stemming from the weaker Mb ligand-duplex interaction at the DNA slide probe interface. The solid-state DNA slide probe based on the G-quadruplex-Mb complex optical probe demonstrated impressive analytical capabilities, including a linear range from 5 × 10-14 to 5 × 10-6 M RNA, a limit of detection (LOD) of 3.51 × 10-15 M, and as low as 1.3 copy number for the positive control plasmid. Clinical sample testing validated its applicability, with the resultant Mb-binding duplex optical amplifier on the silica surface revealing a dynamic linear range for the 2019-nCoV_N positive control plasmid from 2 to 200,000 copies (R2 = 0.9536), conferring a preliminary proof-of-concept study of the DNA slide probe for label-free optosensing of viral RNA. The DNA slide probe, enabling two-dimensional optical transmission/absorption measurements and imaging, provides a promising molecular recognition platform for label- and amplification-free biochemical assays.