Ratiometric Fluorescence Biosensing of Multi-Silver Nanoclusters Hosted in Five-Branched DNA Nanostructure for Detection of Coronavirus DNA Biomarker

Exploring the spectral features of multi-Ag nanoclusters (5rAgNC) populated in five-branched DNA nanostructures (fbDNS) might be very fascinating for label-free ratiometric fluorescence biosensing. Herein, the complementary hybridization was used to self-assemble fbDNS for in situ synthesizing red multi-Ag nanoclusters (5rAgNC). By using fluorescent intensity ratio of 5rAgNC over green Ag nanocluster (gAgNC) as the signal, highly sensitive analytical method for detecting short-stranded DNA biomarker ( I *, 25-nt) related to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) was developed. The specific recognition of I * enabled the sequential dissociation and hybridization for assembling rigid fbDNS within five functional hairpins, thereby displacing I * for repetitive recycling, and their five arms all tethered the same sticking toeholds. The sticking toeholds complement was encoded in the loop of dual-template hairpin (dtH) being able to host gAgNC and 5rAgNC between two conformations. The specific binding made dtH lose its hairpin structure to guide the immobilization in the sticking arms of fbDNS. Resultantly, the structure reconstruction of immobilized dtH released stem segment to connect 5′-toehold bases for 5rAgNC clustering. In a typical reaction route, the rising of I * concentration led to an opposite change with gradual increasing of fluorescent intensity of 5rAgNC and decreasing of fluorescent intensity of gAgNC. Based on this, a sensitive detection method for I * concentration was fabricated. In the concentration range of 5 pmol/L–10 nmol/L, the fluorescence intensity ratio of 5rAgNC to gAgNC had a linear relationship with logarithm of I * concentration. The limit of detection (LOD, 3σ) for I * was 2.9 pmol/L. This method exhibited good specificity for I *. The strategy based on recycled amplification of I * and cooperative emission of multi-nanoclusters displayed superior advantages such as easy operation, fast reaction kinetics, efficient conversion, and improved analysis precision.