A versatile single-molecule counting-based platform by generation of fluorescent silver nanoclusters for sensitive...

The good photostability and strong brightness of individual DNA-templated silver nanoclusters (DNA-AgNCs) have been confirmed by single-molecule imaging in this work and DNA-AgNCs as a new class of outstanding fluorophores are applied in the construction of single-molecule counting-based probes for the first time. Based on the fluorescent AgNC-generating molecular beacons (AgNC-MBs), we present a versatile method for simultaneous analysis of multiple nucleic acids. Distinct from previous designs in which a AgNC stabilizing sequence is incorporated into the stem of a hairpin DNA to form the AgNC-MB, we prepared a nicked MB in which the AgNC stabilizing sequence is hybridized with the longer stem of a single-stranded DNA (ssDNA) with a stem-loop structure. Our proposed AgNC-MB is activated by probe-target hybridization then releasing the AgNC stabilizing sequence via a toehold-mediated strand displacement reaction, the versatility of which has been greatly improved because bases in the target-binding region are not involved in the formation of DNA-AgNCs. As a proof of concept, the simultaneous detection of two breast cancer-related MicroRNAs (miR-21 and let-7a miRNA) has been achieved with total internal reflection fluorescence (TIRF)-based imaging and the detection sensitivity of our method has been demonstrated to be improved by at least two orders of magnitude compared with conventional AgNC-MBs. Furthermore, in the single-nucleotide mutation identification assay, the simultaneous detection strategy introduces a competitive reaction between the two probe-target hybridizations, resulting in the excellent discrimination ability of the AgNC-MB sensing platform and the mutant-type targets can be successfully detected at low abundance. The new AgNC-MB sensing platform demonstrated potential to make AgNCs an attractive alternative to conventional organic dyes for single-molecule studies.